Immunogenic compositions and uses thereof

ABSTRACT

Immunogenic compositions comprising viral vectors and surfactants are provided. Methods for administration and preparation of such compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/907,259, filed Feb. 27, 2018, which is a division of U.S. patentapplication Ser. No. 15/081,601, filed Mar. 25, 2016, which claims thebenefit of U.S. Provisional Patent Application No. 62/137,922, filedMar. 25, 2015, the entirety of each of which is incorporated herein byreference. This application is also related to U.S. patent applicationSer. No. 13/750,774, filed Jan. 25, 2013, the entirety of which isincorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant no. U01AI078045 awarded by National Institutes of Health, NIAID. The governmenthas certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSBPI053USC1_ST25.txt”, which is 2 KB (as measured in MicrosoftWindows®) and was created on Apr. 22, 2019, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND

Vaccination has increased the average human lifespan worldwide more than10 years during the 20th century. Breakthroughs in immunology, molecularbiology and biochemistry in the last 25 years produced more than half ofthe vaccines used during the last 100 years. Despite this, littleprogress has been made in delivery since most are injectable and requirestrict maintenance of cold chain conditions.

Injectable vaccines have various drawbacks. Injections are the mostcommon reason for iatrogenic pain in childhood and deter many fromimmunization Injectable vaccines pose a significant risk to the safetyof medical staff, patients and community. And most vaccines are unstableat ambient temperatures and require refrigeration.

SUMMARY

In a first embodiment, there is provided an immunogenic compositioncomprising a recombinant virus vector (e.g., a recombinant virus vectorcomprising an expression cassette encoding a heterologous antigen), saidrecombinant virus vector formulated in a pharmaceutically acceptablecarrier comprising: (i) PMAL-C16 or (ii) from about 0.1% to 10°% of azwitterionic surfactant. In some aspects, the pharmaceuticallyacceptable carrier comprises PMAL-C16, such as about 0.1 to 50 mg/ml, 1to 40 mg/ml, 1 to 30 mg/ml, 1 to 20 mg/ml, or 5 to 15 mg/ml (e.g., about10 mg/ml) of PMAL-C16. In further aspects, the pharmaceuticallyacceptable carrier comprises about 0.1% to 10%, 0.5% to 10%, 0.5% to5%/0, 10% to 10%, or 1% to 5% of PMAL-C16. In further aspects, thecarrier comprises from about 0.1% to 10%, 0.5% to 10%, 0.5% to 5%, 1% to10%, or 1% to 5% of a zwitterionic surfactant. In particular aspects,the zwitterionic surfactant has a lipid group having a carbon chain of13-30 carbon atoms. In further aspects, the carrier also comprises a pHbuffering agent (e.g., phosphate buffered saline). In certain aspects,the carrier has a pH of between 5.0 and 8.0, between 5.5 and 8.0,between 6.0 and 8.0, between 6.0 and 7.5 or between 6.1 and 7.4. Instill a further aspect, the pharmaceutically acceptable carriercomprises a liquid and comprises between about 1×10⁵ and 1×10¹³, 1×10⁶and 1×10¹³, 1×10⁷ and 1×10¹³, 1×10⁷ and 1×10¹², 1×10⁸ and 1×10¹², 1×10⁹and 1×10¹², or 1×10¹⁰ and 1×10¹³ infectious virus particles (e.g., ofadenovirus) per ml. In yet further aspects, a composition of theembodiments is defined as able to retain at least about 10%, 50%, 70%,80%, 90% or 95% (e.g., 80-95%) of the starting concentration ofinfectious virus after storage at room temperature for 2 months, 4months, 6 months or 8 months.

In a further embodiment there is provided an immunogenic compositioncomprising a recombinant virus vector (e.g., a recombinant virus vectorcomprising an expression cassette encoding a heterologous antigen), saidrecombinant virus vector formulated in a substantially solid carriercomprising: (i) PMAL-C16 or (ii) from about 0.1% to 100% of azwitterionic surfactant. In some aspects, the substantially solidcarrier comprises less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%water. In certain aspects, the substantially solid carrier comprisesPMAL-C16, such as about 0.1 to 50 mg/ml, 1 to 40 mg/ml, 1 to 30 mg/ml, 1to 20 mg/ml, or 5 to 15 mg/ml (e.g., about 10 mg/ml) of PMAL-C16. Infurther aspects, the substantially solid carrier comprises about 0.1% to10%, 0.5% to 10%, 0.5% to 5%, 1% to 10%, or 1% to 5% of PMAL-C16. Infurther aspects, the substantially solid carrier comprises from about0.1% to 10%, 0.5% to 10%, 0.5% to 5%, 1% to 10, or 1% to 5% of azwitterionic surfactant. In particular aspects, the zwitterionicsurfactant has a lipid group having a carbon chain of 13-30 carbonatoms. In further aspects, the carrier also comprises a pH bufferingagent (e.g., phosphate buffered saline). In certain aspects, the carrierhas a pH of between 5.0 and 8.0, between 5.5 and 8.0, between 6.0 and8.0, between 6.0 and 7.5 or between 6.1 and 7.4. In still a furtheraspect, the substantially solid carrier comprises a thin film andcomprises a between about 1×10⁵ and 1×10¹³, 1×10⁶ and 1×10¹³, 1×10⁷ and1×10³, 1×10⁷ and 1×10¹², 1×10⁸ and 1×10¹², 1×10⁹ and 1×10¹², or 1×10¹⁰and 1×10¹³ infectious virus particles (e.g., of adenovirus) per cm³. Inyet further aspects, a composition of the embodiments is defined as ableto retain at least about 10%, 500%, 70%, 80%, 90% or 95% (e.g., 80-95%)of the starting concentration of infectious virus after storage at roomtemperature for 6 months, 12 months, 24 months or 36 months.

In still aspects, a composition of the embodiments further comprises astabilizing agent, such as a sugar, a polymer, amino acids, such asglycine and lysine, or a lyoprotectant. In further aspects, thestabilizing agent comprises a carbohydrate stabilizing agent. Forexample, the stabilizing agent can comprise dextrose, mannose,galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol,pluronic F68, melezitose or mixture thereof.

In some aspects, the recombinant virus vector is a non-enveloped virus,such as a non-enveloped DNA virus. In further aspects, the recombinantvirus vector is an adenovirus vector, such as a vector comprising aE1/E3 deletion. In particular aspects, the adenovirus vector is anadenovirus 5 vector. In yet further aspects the virus is an envelopedvirus (e.g. influenza virus).

A heterologous antigen according to the embodiments can be any ofvariety of antigens, including but not limited to, a cancer cell antigenor an infectious disease antigen, such as a viral, bacterial or parasiteantigen. In certain aspects, the heterologous antigen is a heterologousviral polypeptide, such as a viral envelope polypeptide. For example,the heterologous antigen may be an Ebola virus polypeptide, such as theEbola virus glycoprotein. In some further aspects, the expressioncassette encodes a heterologous antigen, which has been codon optimizedfor expression in mammalian (e.g., human) cells. Additional exemplaryantigens for use according to the embodiments are detailed below.

In a further specific embodiment there is provided an immunogeniccomposition comprising a recombinant adenovirus vector comprising anexpression cassette encoding a heterologous antigen, said recombinantvirus vector formulated in a substantially solid carrier comprising fromabout 0.1% to 10% of a zwitterionic surfactant, said zwitterionicsurfactant having a lipid group with a carbon chain of 13-30 carbonatoms. In a particular aspect, the antigen is an Ebola virusglycoprotein.

In yet a further embodiment, there is provided a method for providing animmune response in a mammal comprising obtaining a composition inaccordance with the embodiments and aspects described above, which hasbeen dispersed in a pharmaceutically acceptable liquid, andadministering an effective amount of the dispersed composition to amammal. In certain aspects, such a method comprises obtaining acomposition in a substantially solid carrier and dispersing thecomposition in a pharmaceutically acceptable liquid (e.g., water). Insome aspects, the administering comprises administering the dispersedcomposition to a mucosal tissue of the mammal. In certain aspects, theadministering is by oral, sublingual, buccal or intranasaladministration. In particular aspects, the pharmaceutically acceptableliquid is water or saline solution. In certain aspects, obtaining thecomposition comprises solubilizing the solid composition in an aqueousliquid such as by contacting the solid with the aqueous liquid andincubating the solid and aqueous liquid for certain period of time,e.g., 1 to 15 minutes.

In yet a further embodiment there is provided a method for providing animmune response in a mammal comprising obtaining a composition arecombinant virus vector (e.g., an adenovirus vector) in apharmaceutically acceptable carrier, said carrier comprising: (i)PMAL-C16 or (ii) from about 0.1% to 10% of a zwitterionic surfactant,and administering an effective amount to the composition to a subject,wherein the subject has been previously exposed to a virus that crossreacts antigenically with the virus vector of the composition. Thus, insome cases, a subject for treatment according to the embodimentscomprises antibodies (e.g., neutralizing antibodies) that bind to therecombinant virus vector. In certain specific aspects, the virus vectoris an adenovirus 5 vector and the subject has been previously exposed toadenovirus 5. In further aspects, the virus (e.g., virus vector) of thecomposition is an influenza virus and the subject has been previouslyexposed to influenza virus. In a further embodiment there is a provideda method for protecting a viral vector from a pre-existing immuneresponse in a subject comprising formulating the viral vector with aneffective amount of a zwitterionic surfactant (e.g., PMAL-C16) andadministering the formulated viral vector to the subject.

In yet still a further embodiment there is provided a method of making astabilized immunogenic composition comprising formulating a solutioncomprising a recombinant virus vector (e.g., an adenovirus vector) in apharmaceutically acceptable carrier, said carrier comprising: (i)PMAL-C16 or (ii) from about 0.1% to 10% of a zwitterionic surfactant,and then drying the solution to provide a stabilized immunogeniccomposition. In certain aspects, drying the solution comprisesdispersing the solution in a thin film and allowing the liquid toevaporate. In further aspects, the method additionally comprisesaliquoting an amount of the stabilized immunogenic composition into acontainer.

In some aspects, prior to drying, the solution comprises about 0.1 to 50mg/ml, 1 to 40 mg/ml, 1 to 30 mg/ml, 1 to 20 mg/ml, or 1 to 10 mg/ml ofthe zwitterionic surfactant. In other aspects, prior to drying, thesolution comprises about 0.1 to 50 mg/ml, 1 to 40 mg/ml, 1 to 30 mg/ml,1 to 20 mg/ml, or 1 to 10 mg/ml of PMAL-C16.

The present disclosure generally relates to vaccine compositions thatmay be administered to a subject via the buccal and/or sublingualmucosa. In some embodiments, the present disclosure also relates tomethods for administration and preparation of such vaccine compositions.

In one embodiment, the present disclosure provides a compositioncomprising an antigen dispersed within an amorphous solid.

In another embodiment, the present disclosure provides a methodcomprising administering a vaccine composition comprising an antigendispersed within an amorphous solid to the buccal and/or sublingualmucosa of a subject in an amount effective to induce an immune responseto the antigen.

In yet another embodiment, the present disclosure provides a methodcomprising providing an antigen and a solution comprising a sugar, sugarderivative or a combination thereof; dispersing the antigen within thesolution to form a mixture; and allowing the mixture to harden so as toform an amorphous solid.

The features and advantages of the present invention will be apparent tothose skilled in the art. While numerous changes may be made by thoseskilled in the art, such changes are within the spirit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Some specific example embodiments of the disclosure may be understood byreferring, in part, to the following description and the accompanyingdrawings. The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIGS. 1A-1C: Multi-Component Formulations Improve AdenovirusTransduction Efficiency and Stabilize Virus in PLGA Microspheres. (1A)Transduction Efficiency of Excipients and Formulations in DifferentiatedCalu-3 Cells. Cell monolayers were exposed to formulations containing amodel recombinant adenovirus serotype 5 vector expressingbeta-galactosidase (AdlacZ) for 2 hours at 37° C. Transductionefficiency was determined by comparison of the number of cellsexpressing the beta-galactosidase transgene after treatment withformulated virus to the number of beta-galactosidase positive cellsafter treatment with virus in saline. Results are reported as themean±standard error of the mean of data generated from triplicatesamples over three separate experiments (n=9 each formulation). PF68,Pluronic F68; nDMPS, N-dodecyl-3-D-maltopyranoside; F3, formulationcontaining sucrose (10 mg/ml), mannitol (40 mg/ml) and 1% (v/v)poly(ethylene) glycol 3,000. *indicates a significant difference withrespect to unformulated virus (1B) Adenovirus Concentration Versus TimeProfiles of Supernatants Collected from PLGA Microspheres Stored at 37°C. Ten milligrams of microspheres containing AdlacZ were suspended in0.5 ml of sterile saline immediately after preparation (ImmediateRelease) or after storage at room temperature (25° C.) for 7 or 30 days.The number of infectious particles released at each time point wasdetermined by serial dilution of collected supernatants and subsequentinfection of Calu-3 cells. (1C) In Vitro Release Profiles of Adenovirusfrom PLGA Microspheres Stored at Room Temperature Over Time. Releaserates for freshly prepared beads did not significantly differ from thoseof beads stored at 25° C. for 7 days. The release rate increasedthreefold after storage for one month under the same conditions. Resultsdepicted in Panels B and C are reported as the mean±standard error ofthe mean of data generated from triplicate samples collected from sixseparate experiments.

FIGS. 2A-2H. Formulations Improve Adenovirus Transduction Efficiency inthe Lungs of Naïve Mice and Those with Prior Exposure to Adenovirus.Naïve C57BL/6 mice were given 5×10¹⁰ particles of the model recombinantvirus used for in vitro screening of formulations (AdlacZ) suspended inpotassium phosphate buffered saline (2A), in formulation F3 (2B),PEGylated virus (2C) or 4.6 mg of PLGA microspheres containing the samedose of virus (2D) by the intranasal route. A second set of mice weredivided into the same treatment groups 28 days after receiving a dose of5×10¹⁰ particles of AdNull, an E1/E3 deleted recombinant adenovirusserotype 5 virus similar to the AdlacZ vector which does not contain atransgene cassette (2E-2H). Mice in each group were sacrificed 4 daysafter administration of the AdlacZ vector. Images display representativegene expression patterns for 6 mice per treatment group. Magnificationin each panel: 200×.

FIGS. 3A-3E: Formulated Preparations Maintain Antigen SpecificPoly-Functional T Cell Responses in Naïve Mice and Those with PriorExposure to Adenovirus. Characterization of the immune response to Ebolaglycoprotein was performed in B10.Br mice as described previously (Patelet al., 2007; Croyle el al., 2008; Choi el al., 2012; Choi et al.,2013). (3A) Magnitude of the Systemic CD8+ T Cell Response Against EbolaGlycoprotein. The number of IFN-γ secreting mononuclear cells wasquantitated in isolates taken 10 days after immunization from the spleenof naïve B10.Br mice and those with prior-exposure to adenovirus byELISpot. (3B) Magnitude of the Mucosal CD8+ T Cell Response AgainstEbola Glycoprotein. The number of IFN-γ secreting mononuclear cells wasquantitated 10 days after immunization in bronchioalveolar lavage (BAL)fluid of naïve mice and those with prior-exposure to adenovirus byELISpot. (3C) Polyfunctionality of the Ebola Glycoprotein-specific TCell Response in Naïve mice. Ten days after immunization, splenocytesfrom 5 mice per treatment group were pooled and stimulated with an Ebolaglycoprotein-specific peptide. Bar graphs illustrate the percentage ofCD8⁺ tumor necrosis factor α (TNF-α)-, interleukin 2 (IL-2)- andinterferon γ (IFN-γ)-producing cells detected after 5 hours of antigenstimulation. Distribution of single-, double- andtriple-cytokine-producing CD8⁺ T cells is shown as various colors in piechart diagrams. The relative frequency of cells that produce all threecytokines defines the quality of the vaccine-induced CD8+ T cellresponse. The proportion of these cells (IFN-γ+IL-2TNF-α) generated inresponse to each treatment is written in the red section of each piechart while the proportion of cells producing a single cytokine arerepresented by the light blue, purple and yellow sections of each piechart. (3D) Polyfunctionality of the Ebola Glycoprotein-Specific T CellResponse in Mice with Prior Exposure to Adenovirus. Pre-existingimmunity to adenovirus 5 was induced by instilling 5×10¹⁰ virusparticles of AdNull, an E1/E3 deleted virus that does not contain atransgene cassette, in the nasal cavity of mice 28 days prior toimmunization. Ten days after immunization, splenocytes were harvestedand pooled as described in 3C. An increase in the number ofpolyfunctional cells, as indicated by an increase in the size of the redsection of each pie graph, was fostered by several of the testformulations with respect to that produced by unformulated vaccine. (3E)Quantitative Analysis of the Effector Memory T Cell Response.Splenocytes were harvested 42 days after immunization, stained with CFSEand stimulated with the TELRTFSI peptide for 5 days. Cells positive forCD8+, CD44^(HI) and CD62L^(LOW) markers were then evaluated for CFSE byfour-color flow cytometry. Data represent the average values obtainedfrom three separate experiments each containing 5 mice per treatment.Error bars reflect the standard error of the data. *p<0.05, **p<0.01,***p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis.

FIGS. 4A-4B: Formulated Vaccines Improve the Anti-Ebola GlycoproteinAntibody Response. Serum collected from individual mice 42 days afterimmunization was screened for total IgG and IgG isotypes by ELISA. (4A)Antibody Profile for Naïve Mice. Naïve B10.Br mice were given 1×10⁸particles of Ad-CAGoptZGP suspended in formulation or 4.6 mg of PLGAmicrospheres containing the virus in KPBS by the intranasal route. (4B)Antibody Profile for Mice with Pre-Existing Immunity to Adenovirus.Pre-existing immunity was established by instillation of a dose of5×10¹⁰ particles of AdNull in the nasal passages of B10.Br mice 28 daysprior to immunization with formulated vaccines. In both panels, theaverage optical density read from samples obtained from each treatmentgroup are presented to serve as a measure of relative antibodyconcentration and data reported as average values±the standard error ofthe mean obtained from three separate experiments each containing 5 miceper treatment. In each panel, the asterisk indicates a significantdifference with respect to naive, immunized animals. *p<0.05, **p<0.01,***p<0.001, one-way ANOVA, Bonferroni/Dunn post-hoc analysis.

FIGS. 5A-5C: Formulations that Augment Both the Polyfunctional T cellResponse and Antigen-Specific IgG1 Antibody Levels in Mice with PriorExposure to Adenovirus Improve Survival from Lethal Challenge. NaïveB10.Br mice were given 1×10⁸ particles of Ad-CAGoptZGP suspended informulation or 4.6 mg of PLGA microspheres containing the virus in KPBSby the intranasal route. Pre-existing immunity (PEI) was established byinstillation of a dose of 5×10¹⁰ particles of AdNull in the nasalpassages of B10.Br mice 28 days prior to immunization. Twenty eight daysafter immunization, mice (n=10/group) were challenged with a lethal doseof 1,000 pfu mouse-adapted Ebola (30,000×LD₅₀) by intraperitonealinjection. (5A) Kaplan-Meier survival curve. * indicates a significantdifference with respect to the PEI/Unformulated treatment group. (5B)Body weight profile after challenge. No significant changes in bodyweight were noted in animals that survived challenge. The mostsignificant drop in weight (˜15% reduction) was observed in animals withprior exposure to adenovirus immunized with the PEGylated preparation.(5C) Serum alanine (ALT) and aspartate (AST) aminotransferase levelspost-challenge. Samples from non-survivors were taken at time of death.Samples from survivors were taken 14 days post-challenge. In all panels,*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA, Bonferroni/Dunn post-hocanalysis.

FIG. 6A-6D: Poly Maleic Anhydrides: Amphiphilic Compounds That ImproveAdenovirus Transduction Efficiency with Minimal Toxicity. A series ofzwitterionic polymers of varying size were screened for their ability toimprove the transduction efficiency of recombinant adenoviruses in lungepithelial cells. Initial screening of formulations in vitro and in vivowas performed with AdlacZ containing the beta-galactosidase transgene(6A and 6D). Use of an E1/E3 deleted recombinant adenovirus expressinggreen fluorescent protein (AdGFP) and quantitation of infected cells byflow cytometry enhanced sensitivity of the screening assay so thatsubtle differences in transduction efficiency in the presence ofanti-adenovirus neutralizing antibodies could be detected (6C). (6A)Transduction Efficiency of Formulated AdlacZ In the Presence ofNeutralizing Antibody. Formulations containing 1×10⁸ infectiousparticles of AdlacZ were incubated with aliquots of a highlycharacterized neutralizing antibody stock for 1 hour prior to infectionof Calu-3 cells. Forty-eight hours later, beta-galactosidase positivecells were identified by histochemical staining. The number ofinfectious virus particles was tallied and calculated as describedpreviously (Callahan et al., 2008). (6B) Toxicity Profile of F16.Formulations were placed on differentiated Calu-3 cell monolayers for aperiod of 2 hours. Culture media was then assessed for LDH activity.Lysis buffer served as a positive control (100% lysis) and KPBS as anegative control. (6C) Quantitative Assessment of TransductionEfficiency of Formulated AdGFP Over a Range of Neutralizing AntibodyConcentrations. In this experiment, 1×10⁸ infectious particles of AdGFPwere incubated in solution containing concentrations of anti-adenovirusantibody reflective of that found in the global population (Barouch etal., 2011; Choi et al., 2012) as described in Panel A. Twenty-four hoursafter infection, infected cells, positive for GFP, were counted by flowcytometery. (6D) Histological Evaluation of Transgene Expression in theLung 4 Days After Intranasal Administration of Formulated Virus. Asingle dose of 5×10¹⁰ infectious particles of AdlacZ was given to naïvemice or mice with PEI to adenovirus induced by the intranasal route.Four days later, mice were sacrificed, tissue harvested and stained fortransgene expression. Sections illustrate representative transgeneexpression patterns found in tissue collected from 6 animals pertreatment. Magnification for Unformulated panels: 200×. Magnificationfor F16 panels: 400×. Results in FIGS. 6A-6C are reported as themean±standard error of the mean of data generated from triplicatesamples collected from four separate experiments.

FIGS. 7A-7C: Formulation F16 Improves Quantitative and Qualitative EbolaGlycoprotein-Specific CD8+ T Cell Responses in Mice with Prior Exposureto Adenovirus. PEI to adenovirus 5 was induced by instilling 5×10¹⁰virus particles of AdNull, an E1/E3 deleted virus that does not containa transgene cassette, in the nasal cavity of mice 28 days prior toimmunization. (7A) The Systemic Effector CD8⁺ T Cell Response. Ten daysafter vaccination, mononuclear cells from the spleen were harvested,stimulated with an Ebola GP-specific peptide and responsive cellsquantitated by ELISpot. (7B) The Mucosal Effector CD8⁺ T Cell Response.Ten days after vaccination, mononuclear cells collected from BAL fluidwere harvested, pooled according to treatment, stimulated with an EbolaGP-specific peptide and responsive cells quantitated by ELISpot. (7C)The Polyfunctional CD8⁺ T Cell Response. Ten days after immunization,splenocytes from 5 mice per treatment group were pooled and stimulatedwith an Ebola glycoprotein-specific peptide. Each positively respondingcell was assigned to one of 7 possible combinations of IFN-γ, IL-2 andTNF-α production and quantitated as shown in the bar graph. The mostpotent responders, those producing all 3 cytokines in response tostimulation, are depicted by the red arcs in the pie charts. Theproportion of cells in samples from each treatment group that produceIFN-γ is depicted by the blue arc. The number in each pie chart denotesthe percentage of triple producers found in samples from a giventreatment group. Data reflect average values±the standard error of themean for six mice per group. *indicates a significant difference withrespect to the Naive/unformulated group, *p<0.05, **p<0.01, one-wayANOVA, Bonferroni/Dunn post-hoc analysis.

FIG. 8. Formulation F16 Improves the Antigen Specific Antibody Responsein Mice with Prior Exposure to Adenovirus. The average optical densityread from individual samples obtained from each treatment group arepresented to serve as a measure of relative antibody concentration anddata reported as average values±the standard error of the mean obtainedfrom two separate experiments each containing 6 mice per treatment. Thelimit of detection for the assay is 0.01 absorbance unit. **p<0.01,one-way ANOVA, Bonferroni/Dunn post-hoc analysis.

FIG. 9: Schematic illustration of vaccination process for liquidformulations and/or those reconstituted from a solid matrix. The sedateanimal's head was rested upon an empty tuberculin syringe to keep thehead in an upright position and to minimize choking or accidentalswallowing of vaccine.

FIG. 10: Timeline and sampling schedule for primate Study 1. Animalswere screened for signs of prior exposure to adenovirus (anti-Ad5 NAB,Ad5 DNA, T cell responses) 4 days prior to immunization. Baseline bloodchemistry panels were also evaluated at this time. Samples were takenfor evaluation of blood chemistry and adenovirus shedding (nasal andoral swabs, urine, feces) 6 h after immunization and on days 1, 2, and7. On day 20, serum and BAL were collected for assessment of sheddingand anti-Ad5 NAB and anti-Ebola GP antibody levels. BAL, PBMCs, and ILNswere also screened for Ebola GP-specific CD8+ and CD4+ T cells at thistime point. On day 38, additional samples were taken for assessment ofanti-Ebola GP and anti-Ad5 antibodies and antigen-specific T cellproliferation (Ebola GP and Ad5). 42 days after immunization, NHPs wereshipped to the National Microbiology Laboratory in Winnipeg, Canada, forchallenge. After an acclimation period, primates were challenged with1,000 pfu of Ebola (1995, Kikwit) by intramuscular (IM) injection.

FIGS. 11A-11D: Primate Study 1: Clinical parameters evaluated over timein non-human primates immunized by various routes. Cynomolgus macaqueswere given a single dose of vaccine by IM injection or by therespiratory or the SL route. Each line represents alterations for eachparameter during the course of therapy for one primate. In each panel:Red lines/squares: saline control. Green lines/circles: IM injection.Blue lines/triangles: IN/IT immunization. Orange lines/diamonds: SLimmunization.

FIGS. 12A-12F: Primate Study 1: Adenovirus genomes are releasedpredominantly in the nasal mucosa and feces after respiratoryimmunization and in the oral and nasal mucosa after sublingualimmunization. Male cynomolgus macaques were given either 1×10⁹ ivp by IMinjection or 1×10¹⁰ ivp by the respiratory or the SL route. DNA wasisolated from each sample, and viral genomes were determined by realtime PCR. Animal numbers and corresponding treatments are outlined inTable 1.

FIGS. 13A-13F: Primate Study 1: Respiratory immunization induces strongantigen-specific T cell responses after administration of a single doseof a formulated adenovirus-based Ebola vaccine. (13A) Quantitativeanalysis of Ebola glycoprotein-specific CD4⁺ T cells in BAL fluid. Cellswere isolated from whole blood 20 days after immunization and stimulatedwith a peptide library for Ebola glycoprotein or peptides specific forthe MHC class II associated invariant chain peptide that binds the MHCclass II groove of cells (h-Clip, negative control). Positive controlcells were stimulated with PMA and ionomycin. Each cell population wasstimulated for 5 h, stained for phenotypic markers, and analyzed by flowcytometry. (13B) Quantitative analysis of Ebola glycoprotein-specificCD8⁺ T cells in BAL fluid. Cells were treated as described for 13A.(13C) Magnitude of the antigen-specific response of mononuclear cellsisolated from whole blood of macaques. PBMCs were isolated 20 days afterimmunization from whole blood and evaluated for IFN-γ secretion afterstimulation with an Ebola GP-specific peptide library by ELISpot. (13D)Magnitude of the antigen-specific response in mononuclear cells isolatedfrom iliac lymph nodes (ILNs) of primates. MNCs were isolated 20 daysafter immunization from ILNs and evaluated for IFN-γ secretion afterstimulation with an Ebola GP-specific peptide library by ELISpot. (13E)Proliferative capacity of Ebola GP-specific T cells collected 38 daysafter immunization of naive primates by various routes. Theproliferative capacity of CD4⁺ (white bars) and CD8⁺ (black bars) Tcells isolated from whole blood was evaluated for each animal bystimulation for 5 days with an Ebola GP-specific peptide library andsubsequent staining for Ki-67, an intracellular marker for proliferation(Gerdes et al., 1983). (13F) Proliferative capacity of adenovirusserotype 5-specific T cells after immunization by various routes. Cellswere isolated from whole blood 38 days after immunization and stimulatedfor 5 days with a first generation adenovirus that does not contain atransgene cassette (AdNull, MOI 1:1,000). The proliferative capacity ofCD4 (white bars) and CD8⁺ (black bars) T cells was determined byintracellular staining for Ki-67. Animal numbers displayed in each paneland their corresponding treatments are summarized in Table 1.

FIGS. 14A-14C: Primate Study 1: Respiratory immunization induces stronganti-Ebola GP and minimal anti-adenovirus antibody responses in serumand BAL fluid. Serum (14A) was collected 20 and 38 days afterimmunization. BAL fluid (14B) was collected 20 days after immunization.These samples were screened for the presence of anti-Ebola GP antibodiesby ELISA. Serum collected on day 20 was also screened foranti-adenovirus 5 NABs using an infectious titer assay (14C). Data in14C is reported as the dilution at which the infectious titer of a firstgeneration adenovirus expressing the beta-galactosidase transgene wasreduced by 50%. In each panel, error bars represent the standard errorof samples assayed in triplicate from each primate for each time point.

FIGS. 15A-15H: Respiratory immunization confers long-term immunity toEbola in naive NHPs. Naive male cynomolgus macaques (see Table 1 forcharacteristics) were challenged 62 days after immunization with alethal dose of 1,000 pfu (1,000 TCID₅₀) of Ebola virus (1995, Kikwit).(15A) Kaplan-Meier survival curve. (15B) Body weight profile afterchallenge. (15C) Thermal analysis of animals during challenge. (15D)Daily clinical scores for each primate using a standard, approvedscoring methodology throughout the challenge. Variations in serum (15E)alanine aminotransferase (ALT), (15F) alkaline phosphatase (ALP), (15G)blood urea nitrogen (BUN), and (15H) platelets (PLT) were noted inanimals that did not survive challenge. Red line: saline control. Greenlines: IM injection. Blue lines: IN/IT immunization. Orange lines: SLimmunization.

FIGS. 16A-16B: Primate Study 2. (16A) Immunization schedule. Eleven malecynomolgus macaques of Chinese origin were immunized according to theschedule depicted in the figure. Animals were shipped to the NationalMicrobiology Laboratory (NML) in Winnipeg 126 days after immunizationfor challenge on day 150 of the study. (16B) Sample collection schemefor Study 2. Animals were screened for signs of prior exposure toadenovirus and Ebola (anti-Ad5 NAB, Ad5 DNA, anti-Ebola GP antibodies) 1week prior to the initiation of the study. Baseline blood chemistrypanels were also evaluated. Samples were taken for evaluation of bloodchemistry and adenovirus shedding (nasal, oral, rectal swabs, urine,feces) 6 h after immunization as well as on days 1, 2, and 7. On day 20,serum and BAL were collected for assessment of shedding, anti-Ad5 NABs,and anti-Ebola GP antibodies. On day 42, additional samples were takenfor assessment of anti-Ebola GP and anti-Ad5 antibodies andantigen-specific T cell proliferation (Ebola GP and Ad5). 150 days afterimmunization, NHPs were shipped to the National Microbiology Laboratoryin Winnipeg for challenge. IN/IT: intranasal/intratracheal. IM:intramuscular. SL: sublingual. PEI, pre-existing immunity.

FIGS. 17A-17D: Primate Study 2: Clinical parameters demonstratingtransient changes after immunization of naive non-human primates andthose with pre-existing immunity to adenovirus immunized by variousroutes. Naive cynomolgus macaques were given a single dose of vaccine bythe respiratory (IN/IT) or the SL routes. A separate group of animalsfirst received a dose of an adenovirus serotype 5 host range mutantvirus 42 days prior to immunization. Each line represents alterationsfor each parameter after immunization for one individual primate. Bluelines/triangles: IN/IT immunization. Black lines/squares: SLimmunization (primates with pre-existing immunity to adenovirus). Orangelines/diamonds: SL immunization (naive primates).

FIGS. 18A-18F: Primate Study 2: Adenovirus genomes are released in theserum and nasal mucosa after IN/IT administration of formulated vaccineand in the oral mucosa after sublingual immunization. Male cynomolgusmacaques were given either 1.6×10⁹ ivp/kg of vaccine in a formulation of10 mg/mL poly(maleic anhydride-alt-1-octadecene) substituted with3-(dimethylamino)propylamine by the respiratory route or 2×10¹⁰ ivp/kgof vaccine in potassium phosphate buffered saline by the SL route. DNAwas isolated from each sample, and viral genomes were determined by realtime PCR. Animal numbers and corresponding treatments are outlined inTable 2.

FIGS. 19A-19D: Primate Study 2: Mucosal immunization elicits diversepopulations of T cells capable of responding to Ebola glycoprotein 150days after treatment. Quantitative analysis of CD4⁺ T cell populationssecreting individual and combinations of cytokines in response toantigen stimulation after IN/IT administration (19A), SL administrationto naive animals (19C), and SL administration to those with pre-existingimmunity to adenovirus (19D). 19B reflects the quantitative analysis ofCD8+ T cell populations after immunization by the IN/IT route. Eachpositively responding cell was assigned to one of 8 possible categoriesreflecting the production of IFN-γ, IL-2, and IL-4 alone or incombination. Pie charts depict the variety of T cell populations foundin each individual animal. CD4⁺ T cells were not found in samplesobtained from primate 808233 (SL immunization). A single CD8⁺ IL-2⁺population was detected in samples from primate 804819 (PEI-SL) and isnot illustrated as a pie chart.

FIGS. 20A-20F: Primate Study 2: Respiratory immunization inducesproduction of antigen-specific antibodies that are sustained over time.Serum was collected from cynomolgus macaques immunized by the IN route(20A) on days 20, 104, and 142 after immunization and analyzed foranti-Ebola GP IgG by ELISA as described (Choi el al., 2013). Serum wasalso collected from naive primates (20C) and those with pre-existingimmunity to adenovirus (20D) on days 20 and 57 after immunization. Thesesamples along with BAL fluid (20B) collected from all primates werescreened for anti-Ebola GP antibodies in the same manner. Serum fromanimals immunized by the IN/IT route (20E) and from animals immunized bythe SL route (20F) was also screened for anti-adenovirus neutralizingantibodies. In each panel, error bars represent the standard error ofsamples assayed in triplicate from each primate for each time point.

FIGS. 21A-21I: Primate Study 2: A single dose of a formulatedadenovirus-based vaccine protects from lethal challenge 150 days afterimmunization. (21A) Kaplan-Meier survival curve. Cynomolgus macaqueswere given a single dose of 1.4×10⁹ ivp of Ad-CAGoptZGP in a formulationcontaining sucrose (10 mg/ml), mannitol (40 mg/ml) and mg/mL poly(maleicanhydride-alt-1-octadecene) substituted with3-(dimethylamino)propylamine, in phosphate buffered saline. Every animalimmunized with this preparation survived lethal challenge. (21B) Bodyweight profiles of immunized animals challenged with Ebola. Animalssuccumbing to infection experienced a change of ±10% of body weightduring the active infection period. (21C) Body temperature of primatesduring challenge. Body temperature declined in each animal duringchallenge with the most dramatic drops observed in animals that were notprotected from infection. (21D) Clinical scores. Primates were observedon a daily basis during the challenge period. Clinical scores wererecorded for each primate by a blinded technician using a standard,approved scoring methodology. (21E) Lymphocyte profiles. Lymphocytes ofsurviving animals recovered from an initial drop 3 days after challengeand remained stable throughout the remainder of the study. (21F) ELISpotanalysis of the cellular immune response in surviving animals 14 daysafter challenge. PBMCs were isolated from whole blood and stimulatedwith a peptide pool spanning the Ebola glycoprotein. (21G) Plateletcounts of primates during challenge. A notable drop in platelets wasobserved in all animals during challenge. (21H) Serum alanineaminotransferase (ALT) levels during challenge. Samples were collectedfrom animals on day 3 and day 14 and at the time of death. (21I) Bloodurea nitrogen (BUN) profile of immunized animals during challenge. Thisparameter remained unchanged in immunized animals that survivedchallenge. Red lines/circles: saline controls. Blue lines/triangles:IN/IT immunization. Orange lines/diamonds: SL immunization. Blacklines/squares: animals with pre-existing immunity to adenovirusimmunized by the SL route.

FIGS. 22A-22F: Anti-Ebola GP antibodies generated by a formulatedadenovirus-based respiratory vaccine are neutralizing while thoseproduced by an unformulated sublingual vaccine are partiallyneutralizing. The neutralizing capacity of antibodies in serum collectedfrom each primate was assessed using a fluorescence neutralization assay(FIGS. 22A, 22C, and 22E). The amount of Ebola virus present in theserum of animals during challenge was determined using a standardinfectious titer assay (FIGS. 22B, 22D, and 22F). In each panel, dataobtained from animals given saline prior to challenge with Ebola areincluded as red symbols and lines for reference. TCID₅₀=median tissueculture infectious dose 50 or the amount of virus that will producepathological change in 50% of cells that are infected in culture. Theseassays were performed under BSL-4 conditions at the NationalMicrobiology Laboratory in Winnipeg.

FIGS. 23A-23B: Biologicals can be stabilized in small, unit dose filmsfor evaluation of potency and bioavailability of protein based, livevirus and bacteria-based vaccines in a variety of animal models and forevaluation of long-term physical stability of vaccines (23A). Severalthousand doses of a given biological substance can be stabilized inlarge films that can be divided into reproducible single-use pieces(23B).

FIGS. 24A-24D: (24A) Porous surface of dried film (3% HPMC/2%sorbitol/0.2% tragacanth gum/PBS) in the absence of virus(Magnification: 75,000×). (24B) Electron micrograph of dried film (1.5%HPMC/2% Sorbitol/0.2% tragacanth gum/PBS) in the absence of virus(Magnification: 25,000×). (24C) Large Non-Crystalline Pockets in Filmmade of 1.5% HPMC/2% Sorbitol/0.2% tragacanth gum in PBS which FosterStabilization of Virus Particles in the Amorphous State. (Magnification20,000×). (24D) Adenovirus Particles (arrows) Suspended in Film. Thepresence of the virus notably changes the physical characteristics ofthe film as it assumes a non-porous, amorphous shape. Formulation issame as that in 24C. (Magnification 20,000×).

FIG. 25: Films Retain 3 Dimensional Shape of Embedded Virus After 12months of Storage at Room Temperature. Virus particles (70 nm, shadedareas, arrows) embedded in film (Formulation 2 in FIG. 2) and stored inthe dry state for one year. Film was embedded in epoxy resin, sectionedfrozen and transferred directly to the electron microscope under osmiumvapor. (Magnification 20,000×).

FIG. 26: Infectious Enveloped and Non-Enveloped Viruses Can Be Recoveredfrom Dried Film. Infectious titers of recombinant adenovirus expressingthe Ebola Virus glycoprotein (a non-enveloped virus) and PR8 (HINIinfluenza) were evaluated in liquid formulations, dried andreconstituted 48 hours after storage in the dry state at 20° C. Data isrecorded as the difference in titers of each preparation prior to dryingand after reconstitution.

FIG. 27: Solvent System Influences Changes in Film pH During the DryingProcess. The pH of each formulation in the liquid (pre-dry) and drystate was recorded according to the method described in Croyle et al.(2001) Gene Ther. 8: 1281-1290. Prior to drying, 10 microliters ofUniversal pH Indicator Solution (Fisher Scientific) was added to eachformulation and the pH visually recorded. When drying was complete,films were visually inspected and the pH compared to pre-drying values.On the x-axis, 1 is distilled, deionized water, 2 is 120 mM PBS(phosphate buffered saline), and 3 is 10 mM Tris(Tris(hydroxymethyl)aminomethane). Formulations evaluated in this studyconsisted of 0.1-15% hydroxypropyl methylcellulose, 0.1-0.8% tragacanthgum, 1-5% sorbitol and 1-100 mg/ml melezitose.

FIGS. 28A-28C: Solvent System Dictates Recovery of Virus from DriedFilm. Three different aqueous solvent systems were utilized informulations containing: Low (Base 1, 0.5%) (28A), Medium (Base 2, 1.5%)(28B), and High (Base 3, 3%) (28C) concentrations of hydroxypropylmethylcellulose, (Base in figure). Films were dried at ambienttemperature and pressure for 5 hours. Twenty four hours later, each filmwas reconstituted with sterile saline and infectious titer of virusembedded in the preparation determined by serial dilution, infection ofHeLa cells and visual tallying of cells staining positive for virus.Percent recovery was calculated using the following formula:

${\%\mspace{14mu}{Recovery}} = {\frac{\log\left( {{{Infectious}\mspace{14mu}{Titer}\mspace{14mu}{at}\mspace{14mu} t} = 1} \right)}{\log\left( {{{Infectious}\mspace{14mu}{Titer}\mspace{14mu}{at}\mspace{14mu} t} = 0} \right)} \times 100}$

FIG. 29: The pH of the Dried Film Significantly Impacts Recovery ofInfectious Virus After Reconstitution. Recombinant adenovirus was placedin a variety of formulations that were dried as thin films. Twenty-fourhours later, films were reconstituted and viral titer assessed by astandard limiting dilution assay. Data was grouped according to thefinal pH of the dried film. (correlation coefficient r²=0.996)

FIG. 30: Detergent Prevents Drop in Film pH After Drying. The pH offormulations consisting of 0.1-15% hydroxypropyl methylcellulose,0.1-0.8% tragacanth gum, 1-5% sorbitol and 1-100 mg/ml melezitose,without detergent (BASE) and that containing 10 mg/ml PMCAL C16(BASE+DET) in the liquid (pre-dry) and dry state was recorded accordingto the method described in Croyle et al. (2001) Gene Ther. 8: 1281-1290.Prior to drying, 10 microliters of Universal pH Indicator Solution(Fisher Scientific) was added to each formulation and the pH visuallyrecorded. When drying was complete, films were visually inspected andthe pH compared to pre-drying values.

FIGS. 31A-31B: Formulation Dictates Recovery of Virus from Dried Film inCertain Solvent Systems. Three different concentrations of hydroxypropylmethylcellulose (0.5, 1.5 and 3%) were evaluated for their ability toretain infectious titer of virus after drying in 120 mM PBS (Solvent 2)or 10 mM Tris buffer. Films were dried at ambient temperature andpressure for 6 hours. Twenty four hours later, each film wasreconstituted with sterile saline and infectious titer of virus embeddedin the preparation determined by serial dilution, infection of HeLacells and visual tallying of cells staining positive for virus. Percentrecovery was calculated using formula provided above.

FIG. 32: Detergent Significantly Improves Recovery of Infectious Virusfrom Films. Recombinant adenovirus (1.25×10¹² particles) was formulatedin: (A) 0.5% w/w hydroxypropyl methylcellulose containing 2% w/wsorbitol (Formulation 1) or 2% v/v glycerol (Formulation 2); (B) 1.5%w/w hydroxypropyl methylcellulose containing 2% w/w sorbitol(Formulation 3) or 2% v/v glycerol (Formulation 4); (C) 3% w/whydroxypropyl methylcellulose containing 2% w/w sorbitol (Formulation 5)or 2% v/v glycerol (Formulation 6) with (+DET) or without (−DET) 10mg/ml PMAL C16. Formulations were dried into thin films, reconstituted24 hours after drying and infectious titer determined by limitingdilution on HeLa cells.

FIG. 33: The Amount of Virus Embedded in Film Formulation Does notImpact Recovery. Adenovirus particles were incorporated in thin films inamounts ranging from those found post-purification (1×10¹³, 1×10¹²infectious particles) to those which reflect reasonable doses forimmunization and therapeutic purposes (1×10¹¹-1×10⁷ infectiousparticles). These concentrations did not seem to impact recovery fromdried films suggesting that they can be utilized for stabilizingbiologicals during holding steps of manufacturing processes as well asfor single use films for self therapy/immunization.

FIG. 34: Binding Agents Improve Recovery of Recombinant Virus from Film.Inclusion of a binding agent (in this case 2% w/w sorbitol, Binder) inthe film formulation improved recovery of virus by 43% (1.5% HPMC, Base2) and 21.1% (3% HPMC, Base 3) after drying. Addition of plasticizers(in this case 2% v/v glycerol, Plasticizer) did not improve recovery tothe same degree (32.3%, Base 2, 14.1%, Base 3).

FIG. 35: Virus Significantly Impacts the Dissolution Rate of Films inSimulated Saliva. Films of uniform weight containing 1.25×10¹² particlesof recombinant adenovirus (VIRUS) and blank controls were placed insimulated human salivary fluid at 37° C. under gentle stirring.Dissolution rate was calculated by dividing the starting weight of thefilm by the time at which the film could no longer be visibly detectedin the solvent. Simulated human saliva fluid consisted of: KCl 0.15 g/L,NaCl 0.12 g/L, Sodium Bicarbonate 2.1 g/L, alpha-amylase 2.0 g/L andgastric mucin 1.0 g/L as described in Davis et al. (1971) J. Pharm. Sci.60(3):429-432. Formulations included in this study consisted of: (A)0.50% w/w hydroxypropyl methylcellulose containing 2% w/w sorbitol(Formulation 1) or 2% v/v glycerol (Formulation 2); (B) 1.5% w/whydroxypropyl methylcellulose containing 2% w/w sorbitol (Formulation 3)or 2% v/v glycerol (Formulation 4); (C) 3% w/w hydroxypropylmethylcellulose containing 2% w/w sorbitol (Formulation 5) or 2% w/vglycerol (Formulation 6).

FIG. 36: PMAL C16 Significantly Improves Dissolution Time for Films.Films of uniform weight were placed in sterile saline at 37° C. undergentle stirring. Dissolution rate was calculated by dividing thestarting weight of the film by the time at which the film could nolonger be visibly detected in the solvent. Formulations included in thisstudy consisted of: (A) 0.5% w/w hydroxypropyl methylcellulosecontaining 2% w/w sorbitol (Formulation 1) or 2% v/v glycerol(Formulation 2); (B) 1.5% w/w hydroxypropyl methylcellulose containing2% w/w sorbitol (Formulation 3) or 2% v/v glycerol (Formulation 4); (C)3° % w/w hydroxypropyl methylcellulose containing 2% w/w sorbitol(Formulation 5) or 2% w/v glycerol (Formulation 6).

FIG. 37: Film Formulations with and without PMAL C16 Protect AdenovirusFrom Degradation in Saliva. Infectious titer of unformulated virus (AdUnform.) placed in simulated human salivary fluid for 5 minutes droppedby a factor of 3 when compared to the same concentration of virusincubated in sterile saline (Ad Control). The infectious titer of virusformulated in standard film base (1.5% HPMC, FILM) significantlyimproved the titer of the virus in simulated human saliva six-fold. Theaddition of PMAL C16 enhanced protection of the virus from digestiveamylase and mucin. The infectious titer of this preparation (FILM+DET)was 22 times that of the unformulated virus (Ad Unform.).

FIG. 38: Virus Significantly Impacts the Moisture Retained in Dried FilmFormulations. Moisture content for films of uniform weight containing1.25×10¹² particles of recombinant adenovirus (VIRUS) and blank controlswas assessed by Karl Fischer titration according to USP standards.Formulations included in this study consisted of: (A) 0.5% w/whydroxypropyl methylcellulose alone (Formulation1) or containing 2% w/wsorbitol (Formulation 2) or 2% v/v glycerol (Formulation 3); (B) 1.5%w/w hydroxypropyl methylcellulose alone (Formulation 4) or containing 2°% w/w sorbitol (Formulation 5) or 2% v/v glycerol (Formulation 6); (C)3% w/w hydroxypropyl methylcellulose alone (Formulation 7) containing 2%w/w sorbitol (Formulation 8) or 2% v/v glycerol (Formulation 9).

FIG. 39: PMAL C16 Profoundly Increases Moisture Content of FilmsContaining Virus. Moisture content for films of uniform weightcontaining 1.25×10¹² particles of recombinant adenovirus in baseformulation (BASE) and films including detergent (DET) was assessed byKarl Fischer titration according to USP standards. (A) 0.5% w/whydroxypropyl methylcellulose alone (Formulation 1) or containing 2% w/wsorbitol (Formulation 2) or 2% v/v glycerol (Formulation 3); (B) 1.5%w/w hydroxypropyl methylcellulose alone (Formulation 4) or containing 2%w/w sorbitol (Formulation 5) or 2% v/v glycerol (Formulation 6); (C) 3%w/w hydroxypropyl methylcellulose alone (Formulation 7) containing 2%w/w sorbitol (Formulation 8) or 2% v/v glycerol (Formulation 9)

FIG. 40: Recombinant Adenovirus Can Be Evenly Distributed Across LargeFilm that can be Divided into Equal Unit Doses. A 3 cm×3 cm filmcontaining recombinant adenovirus was dried and divided into nine 1 cm×1cm parts (black grid, lower right corner of plot). Each part wasreconstituted with sterile saline and titer assessed by an in vitroassay. Data shown is representative of 3 different formulations.Formulations included in this study consisted of: 0.5% w/w hydroxypropylmethylcellulose containing 2% w/w sorbitol (Formulation 1), 1.5% w/whydroxypropyl methylcellulose containing 2% w/w sorbitol (Formulation 2)and 3° % w/w hydroxypropyl methylcellulose containing 2% w/v glycerol(Formulation 3). Formulations were prepared in 120 mM PBS.

FIGS. 41A-41C: Formulations can significantly extend shelf-life ofrecombinant adenovirus at ambient temperature in dried and reconstitutedfilms. 41A: 30 month Stability Profile for Ebola Vaccine in Solid FilmMatrix. 41B: 8 month Stability Profile of Ebola Vaccine Reconstitutedfrom Film Matrix and Stored in Liquid Form. Each preparation was storedat 20° C. This is markedly better than the stability seen afterreconstitution of a stable lyophilized formulation of the virus andsubsequent storage at 4° C. (41C).

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as illustrated, in part, by the appendedclaims.

DESCRIPTION

The present disclosure generally relates to vaccine compositions thatmay be administered to a subject via the buccal and/or sublingualmucosa. In some embodiments, the present disclosure also relates tomethods for administration and preparation of such vaccine compositions.

The buccal and the sublingual mucosa are attractive for the delivery ofmedicinal compounds and have largely been uninvestigated in the contextof protective immunization. The sublingual and the buccal epithelium arehighly vascularized, allowing direct entry into the systemiccirculation, avoiding pre-systemic metabolism of antigen in thegastrointestinal tract They harbor a dense lattice of professionalantigen presenting cells (APCs), contain many T lymphocytes and directlyaccess mucosal-associated lymphoid tissues. One of the many advantagesof the present disclosure, many of which are not discussed herein, isthat a vaccine composition of the present disclosure may be administeredby direct application to the cheek (buccal) or under the tongue(sublingual), which may then induce a strong protective systemic andmucosal immune response. Furthermore, in those embodiments where thevaccine is a recombinant adenovirus (“Ad”)-based vaccine, it may beadministered via the buccal and/or sublingual mucosa with significantpotential for successful vaccination of those with pre-existing immunityto Ad5. Pre-existing immunity to Ad5 is a global phenomenon and iscurrently the most significant limitation to the use of these vectors.

The buccal and sublingual mucosa contain an immobile expanse of smoothmuscle upon which of a variety of dosage forms such as lozenges, gels,patches and films can reside (Pather, 2008). This supports an epitheliumof 40-50 layers of actively dividing squamous, non-keratinized cells(Wertz, 1991). Although this layer is the most significant barrier tothe absorption of large molecules though the cheek, cell turnover isslow (4-14 days), allowing for continued release of antigen (Hill,1984). Reagents that aid absorption of large molecules across the mucosa(surfactants, cyclodextrins, polyacrlyates) and polymers that facilitateinteraction with the surface (polycarbophil, carboxymethyl cellulose)also protect labile molecules from degradation at ambient temperatures(Hassan, 2010, Shojaei, 1998). Accordingly, the present disclosure isalso innovative in that it promotes a delivery method that could improvevaccine potency and physical stability at ambient temperatures.

In some embodiments, the present disclosure provides a vaccinecomposition comprising an antigen dispersed within an amorphous solid.As used herein, the term “antigen” means a substance that induces aspecific immune response in a host animal. The antigen may comprise awhole organism, killed, attenuated or live (including killed, attenuatedor inactivated bacteria, viruses, fungi, parasites, prions or othermicrobes); a subunit or portion of an organism; a recombinant vectorcontaining an insert with immunogenic properties; a piece or fragment ofDNA capable of inducing an immune response upon presentation to a hostanimal or whcich contains the genetic material that allows expression ofa given antigen in cells that take up the DNA; a protein, a polypeptide,a peptide, an epitope, a hapten, or any combination thereof.Alternatively, the antigen may comprise a toxin or antitoxin.

In general, an amorphous solid suitable for use in the presentdisclosure should be dissolvable upon contact with an aqueous liquid,such as saliva. In some embodiments, amorphous solids suitable for usein the present disclosure may be formed from any sugar, sugar derivativeor combination of sugars/derivatives so long as the sugar and/orderivative is prepared as a liquid solution at a concentration thatallows it to flow freely when poured but also forms an amorphous phaseat ambient temperatures on a physical surface that facilitates thisprocess, such as aluminum or Teflon. Examples of suitable sugars mayinclude, but are not limited to glucose, dextrose, fructose, lactose,maltose, xylose, sucrose, corn sugar syrup, sorbitol, hexitol, maltilol,xylitol, mannitol, melezitose, raffinose, and a combination thereof.While not being bound to any particular theory, it is believed thatsugars minimize interaction of the antigen with water during storage anddrying, in turn, preventing damage to the three dimensional shape of theantigen due to crystal formation during the drying process andsubsequent loss of efficacy. An example of the surface characteristicsof an amorphous solid is illustrated in FIGS. 23A and 24D In someembodiments, an amorphous solid suitable for use in the presentdisclosure may have a thickness of about 0.05 micrometers to about 5millimeters.

In addition, in some embodiments, certain sugars may also function as abinder which may provide “substance” to pharmaceutical preparations thatcontain small quantities of very potent medications for ease ofhandling/administration They may also hold components together orpromote binding to surfaces (like the film backing) to ease drugdelivery and handling. Lastly, they may also contribute to the overallpharmaceutical elegance of a preparation by forming uniform glasses upondrying.

In certain embodiments, the vaccine compositions of the presentdisclosure also may comprise a water-soluble polymer including, but notlimited to, carboxymethyl cellulose, carboxyvinyl polymers, high amylosestarch, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, methylmethacrylate copolymers, polyacrylic acid,polyvinyl alcohol, polyvinyl pyrrolidone, pullulan, sodium alginate,poly(lactic-co-glycolic acid), poly(ethylene) oxide,poly(hydroxyalkanoate) and a combination thereof.

Furthermore, in some embodiments, the vaccine compositions of thepresent disclosure may further comprise one or more oils, polyalcohols,surfactants, permeability enhancers, and/or edible organic acids.Examples of suitable oils may include, but are not limited to,eucalyptol, menthol, vacrol, thymol, methyl salicylate, verbenone,eugenol, gerianol and a combination thereof: Examples of suitablepolyalcohols may include, but are not limited to, glycerol, polyethyleneglycol, propylene glycol, and a combination thereof. Examples ofsuitable edible organic acids may include, but are not limited to,citric acid, malic acid, tartaric acid, fumaric acid, phosphoric acid,oxalic acid, ascorbic acid and a combination thereof. Examples ofsuitable surfactants may include, but are not limited to, difunctionalblock copolymer surfactants terminating in primary hydroxyl groups, suchas Pluronic® F68 commercially available from BASF, poly(ethylene) glycol3000, dodecyl-3-D-maltopyranoside, disodium PEG-4 cocamidoMIPA-sulfosuccinate (“DMPS”), etc. It is believed that certainsurfactants may minimize interaction of the antigen with itself andother antigens and subsequent formation of large aggregated particlesthat cannot effectively enter and be processed by target and antigenpresenting cells They may also be capable of weakening cell membraneswithout causing permanent damage and, through this mechanism, promoteuptake of large particles though rugged biological membranes such as thebuccal mucosa.

In certain preferred aspects, an immunogenic composition of theembodiments comprises a zwitterionic surfactant. In some embodiments,the zwitterionic surfactant is a surfactant molecule which contains agroup which is capable of being positively charged and a group which iscapable of being negatively charged. In some embodiments, both thepositively charged and negatively charged groups are ionized atphysiological pH such that the molecule has a net neutral charge. Insome embodiments, the positively charged group comprises a protonated orquaternary ammonium. In some embodiments, the negatively charged groupcomprises a sulfate, a phosphate, or a carboxylate. The zwitterionicsurfactant further comprises one or more lipid groups consistingessentially of an alkyl, cycloalkyl, or alkenyl groups. Preferably, thezwitterionic surfactant comprises one or more lipid groups consistingessentially of an alkyl, cycloalkyl, or alkenyl groups with a carbonchain of more than 12 carbon atoms. In some embodiments, the lipid grouphas a carbon chain of 12-30 carbon atoms. In some embodiments, the lipidgroup has a carbon chain of 12-24 carbon atoms. In some embodiments, thelipid group has from 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, to24 carbons, or any range derivable thereof. In some embodiments, thezwitterionic surfactant is a polymeric structure which contains multiplezwitterionic groups and multiple lipid groups on a central backbone. Insome embodiments, the zwitterionic surfactant is a polymer which hasfrom about 50 to about 200 repeating units wherein each repeating unitscomprises one positively charged group, one negatively charged group,and one lipid group. In some embodiments, the zwitterionic surfactant isa polymer which has a 75 to 150 repeating units. In some embodiments,the central backbone is an alkyl, polyethylene glycol, or polypropylenechain. In some embodiments, the central chain is an alkyl group.

Some non-limiting examples of zwitterionic surfactants include3-(N,N-Dimethyltetradecylammonio)propanesulfonate (SB3-14),3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate(C7BzO), 3-(decyldimethylammonio) propanesulfonate inner salt (SB3-10),3-(dodecyldimethylammonio) propanesulfonate inner salt (SB3-12),3-(N,N-dimethyloctadecylammonio) propanesulfonate (SB3-18),3-(N,N-dimethyl-octylammonio) propanesulfonate inner salt (SB3-8),3-(N,N-dimethylpalmitylammonio) propanesulfonate (SB3-16),3-[N,N-dimethyl(3-myristoylaminopropyl)ammonio]propane-sulfonate(ASB-14), CHAPS, CHAPSO, acetylated lecithin, alkyl(C12-30)dialkylamine-N-oxide apricotamidopropyl betaine, babassuamidopropylbetaine, behenyl betaine, bis 2-hydroxyethyl tallow glycinate, C12-14alkyl dimethyl betaine, canolamidopropyl betaine, capric/caprylicamidopropyl betaine, capryloamidopropyl betaine, cetyl betaine,3-[(Cocamidoethyl)dimethylammonio]-2-hydroxypropanesulfonate,3-[(Cocamidoethyl)dimethyl-ammonio]propanesulfonate, cocamidopropylbetaine, cocamidopropyl dimethylamino-hydroxypropyl hydrolyzed collagen,N-[3-cocamido)-propyl]-N,N-dimethyl betaine, potassium salt,cocamidopropyl hydroxysultaine, cocamidopropyl sulfobetaine,cocaminobutyric acid, cocaminopropionic acid, cocoamphodipropionic acid,coco-betaine, cocodimethylammonium-3-sulfopropylbetaine,cocoiminodiglycinate, cocoiminodipropionate, coco/oleamidopropylbetaine, cocoyl sarcosinamide DEA, DEA-cocoamphodipropionate,dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine,N,N-dimethyl-N-lauric acid-amidopropyl-N-(3-sulfopropyl)-ammoniumbetaine, N,N-dimethyl-N-myristyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-palmityl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-stearamidopropyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-stearyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-tallow-N-(3-sulfopropyl)-ammonium betaine, disodiumcaproamphodiacetate, disodium caproamphodipropionate, disodiumcapryloamphodiacetate, disodium capryloamphodipropionate, disodiumcocoamphodiacetate, disodium cocoamphodipropionate, disodiumisostearoamphodipropionate, disodium laureth-5 carboxyamphodiacetate,disodium lauriminodipropionate, disodium lauroamphodiacetate, disodiumlauroamphodipropionate, disodium octyl b-iminodipropionate, disodiumoleoamphodiacetate, disodium oleoamphodipropionate, disodiumPPG-2-isodeceth-7 carboxyamphodiacetate, disodium soyamphodiacetate,disodium stearoamphodiacetate, disodium tallamphodipropionate, disodiumtallowamphodiacetate, disodium tallowiminodipropionate, disodiumwheatgermamphodiacetate,N,N-distearyl-N-methyl-N-(3-sulfopropyl)-ammonium betaine,erucamidopropyl hydroxysultaine, ethylhexyl dipropionate, ethylhydroxymethyl oleyl oxazoline, ethyl PEG-15 cocamine sulfate,hydrogenated lecithin, hydrolyzed protein, isostearamidopropyl betaine,3-[(Lauramidoethyl)dimethylammonio]-2-hydroxypropanesulfonate,3-[(Lauramidoethyl)dimethylammonio]propanesulfonate, lauramido-propylbetaine, lauramidopropyl dimethyl betaine, lauraminopropionic acid,lauroamphodipropionic acid, lauroyl lysine, lauryl betaine, laurylhydroxysultaine, lauryl sultaine, linoleamidopropyl betaine,lysolecithin, milk lipid amidopropyl betaine, myristamidopropyl betaine,octyl dipropionate, octyliminodipropionate,n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate,n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-octadecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate, oleamidopropylbetaine, oleyl betaine, 4,4(5H)-oxazoledimethanol, 2-(heptadecenyl)betaine, palmitamidopropyl betaine, palmitamine oxide, PMAL-C6,PMAL-C12, PMAL-C16, ricinoleamidopropyl betaine, ricinoleamidopropylbetaine/IPDI copolymer, sesamidopropyl betaine, sodium C12-15alkoxypropyl iminodipropionate, sodium caproamphoacetate, sodiumcapryloamphoacetate, sodium capryloamphohydroxypropyl sulfonate, sodiumcapryloamphopropionate, sodium carboxymethyl tallow polypropylamine,sodium cocaminopropionate, sodium cocoamphoacetate, sodiumcocoamphohydroxypropyl sulfonate, sodium cocoamphopropionate, sodiumdicarboxyethyl cocophosphoethyl imidazoline, sodium hydrogenated tallowdimethyl glycinate, sodium isostearoamphopropionate, sodiumlauriminodipropionate, sodium lauroamphoacetate, sodiumoleoamphohydroxypropylsulfonate, sodium oleoamphopropionate, sodiumstearoamphoacetate, sodium tallamphopropionate, soyamidopropyl betaine,stearyl betaine,3-[(Stearamidoethyl)dimethylammonio]-2-hydroxypropanesulfonate,3-[(Stearamidoethyl)-dimethylammonio]propanesulfonate, tallowamidopropylhydroxysultaine, tallowamphopoly-carboxypropionic acid, trisodiumlauroampho PG-acetate phosphate chloride, undecylenamidopropyl betaine,and wheat germamidopropyl betaine.

A vaccine composition of the present disclosure further comprises anantigen. Antigens suitable for use in the present disclosure may includeany antigen for which cellular and/or humoral immune responses aredesired, including antigens derived from viral, bacterial, fungal andparasitic pathogens and prions that may induce antibodies, T-cell helperepitopes and T-cell cytotoxic epitopes. Such antigens include, but arenot limited to, those encoded by human and animal viruses and cancorrespond to either structural or non-structural proteins. Furthermore,the present disclosure contemplates vaccines made using antigens derivedfrom any of the antigen sources discussed below and those that use thesesources as potential delivery devices or vectors. For example, in onespecific embodiment, recombinant adenovirus may be used to deliver Ebolaantigens for immunization against Ebola infection.

Antigens useful in the present disclosure may include those derived fromviruses including, but not limited to, those from the familyArenaviridae (e.g., Lymphocytic choriomeningitis virus), Arterivirus(e.g., Equine arteritis virus), Astroviridae (Human astrovirus 1),Birnaviridae (e.g., Infectious pancreatic necrosis virus, Infectiousbursal disease virus), Bunyaviridae (e.g., California encephalitis virusGroup), Caliciviridae (e.g., Caliciviruses), Coronaviridae (e.g., Humancoronaviruses 299E and OC43), Deltavirus (e.g., Hepatitis delta virus),Filoviridae (e.g., Marburg virus. Ebola virus). Flaviviridae (e.g.,Yellow fever virus group, Hepatitis C virus), Hepadnaviridae (e.g.,Hepatitis B virus), Herpesviridae (e.g., Epstein-Bar virus,Simplexvirus. Varicellovirus, Cytomegalovirus, Roseolovirus,Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g., InfluenzavirusA, B, and C), Papovaviridae (e.g., Papillomavirus), Paramyxoviridae(e.g., Paramyxovirus such as human parainfluenza virus 1, Morbillivirussuch as Measles virus, Rubulavirus such as Mumps virus, Pneumovirus suchas Human respiratory syncytial virus), Picornaviridae (e.g., Rhinovirussuch as Human rhinovirus 1A, Hepatovirus such Human hepatitis A virus,Human poliovirus, Cardiovirus such as Encephalomyocarditis virus,Aphthovirus such as Foot-and-mouth disease virus O, Coxsackie virus),Poxyiridae (e.g., Orthopoxvirus such as Variola virus or monkeypoxvirus). Reoviridae (e.g., Rotavirus such as Groups A-F rotaviruses),Retroviridae (Primate lentivirus group such as human immunodeficiencyvirus 1 and 2), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g.,Rubivirus such as Rubella virus), Human T-cell leukemia virus, Murineleukemia virus, Vesicular stomatitis virus, Wart virus, Blue tonguevirus. Sendai virus, Feline leukemia virus, Simian virus 40, Mousemammary tumor virus, Dengue virus, HIV-1 and HIV-2, West Nile, H1N1,SARS, 1918 Influenza, Tick-borne encephalitis virus complex (Absettarov,Hanzalova, Hypr), Russian Spring-Summer encephalitis virus.Congo-Crimean Hemorrhagic Fever virus, Junin Virus, Kumlinge Virus,Marburg Virus. Machupo Virus, Kyasanur Forest Disease Virus, LassaVirus, Omsk Hemorrhagic Fever Virus, FIV, SIV, Herpes simplex 1 and 2,Herpes Zoster, Human parvovirus (B19), Respiratory syncytial virus, Poxviruses (all types and serotypes), Coltivirus, Reoviruses—all types,and/or Rubivirus (rubella).

Antigens useful in the present disclosure may include those derived frombacteria including, but not limited to, Streptococcus agalacaae,Legionella pneumophilia. Streptococcus pyogenes, Escherichia coli,Neisseria gonorrhosae, Neisseria meningitidis, Pneumococcus, Hemophilisinfluenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonasaeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacteriumtuberculosis, Plasmodium falciparum, Plasmodium vivax, Toxoplasmagondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosomarhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosomajapanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus,Leishmania tropica, Trichinella spiralis, Theileria parva, Taeniahydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus,Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M.arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Candidaalbicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioidesimmitis, Blastomyces dermatitidis, Aspergillus fumigatus, Penicilliummarneffei, Bacillus anthracis, Bartonella, Bordetella pertussis,Brucella—all serotypes, Chlamydia trachomatis, Chlamydia pneumoniae,Clostridium botulinum—anything from clostridium serotypes, Haemophilusinfluenzae, Helicobacter pylori, Klebsiella—all serotypes,Legionella—all serotypes, Listeria, Mycobacterium—all serotypes,Mycoplasma—human and animal serotypes, Rickettsia—all serotypes,Shigella—all serotypes, Staphylococcus aureus, Streptcoccus—S.pneumoniae, S. pyogenes, Vibrio cholera, Yersinia enterocolitica, and/orYersinia pestis.

Antigens useful in the present disclosure may include those derived fromparasites including, but not limited to, Ancylostoma human hookworms,Leishmania—all strains, Microsporidium, Necator human hookworms,Onchocerca filarial worms, Plasmodium—all human strains and simianspecies, Toxoplasma—all strains, Trypanosoma—all serotypes, and/orWuchereria bancrofti filarial worms.

In another embodiment, an antigen is an aberrant protein derived from asequence which has been mutated. Such antigens may include thoseexpressed by tumor cells or aberrant proteins whose structure orsolubility leads to the formation of an aggregation-prone product andcause disease. Examples of aberrant proteins may include, but are notlimited to, Alzheimer's amyloid peptide, SOD1, presenillin 1 and 2,α-synuclein, amyloid A, amyloid P, CFTR, transthyretin, amylin,lysozyme, gelsolin, p53, rhodopsin, insulin, insulin receptor,fibrillin, α-ketoacid dehydrogenase, collagen, keratin, PRNP,immunoglobulin light chain, atrial natriuretic peptide, seminal vesicleexocrine protein, β2-microglobulin, PrP, precalcitonin, ataxin 1, ataxin2, ataxin 3, ataxin 6, ataxin 7, huntingtin, androgen receptor,CREB-binding protein, dentaorubral pallidoluysian atrophy-associatedprotein, maltose-binding protein, ABC transporter,glutathione-S-transferase, and thioredoxin.

In one embodiment, a vaccine composition comprising an amorphous solidmay be made by preparing a solution comprising a sugar, sugar derivativeor combination of sugars/derivatives in a buffer and optionally otheradditives previously mentioned. In some embodiments, a sugar, sugarderivative or combination of sugars/derivatives may be present in thesolution in an amount up to about 60% by weight of the solution. In someembodiments, an additive may be present in an amount of about 5% or lessby weight of the solution. In general, the solution comprising thesugar, sugar derivative or combination of sugars/derivatives is made ata concentration higher than the desired final concentration tocompensate for any dilution that may occur when the antigen is added.The desired antigen may be added to the solution at a concentrationknown to induce the desired immune response. The mixture may then bestirred at ambient temperature until a substantially homogeneous mixtureis obtained. In some embodiments, the mixture may then be brieflysonicated under cooled conditions, e.g. 4° C., to remove any air bubblesthat may have developed. In other embodiments, the mixture may beslightly heated, e.g., heated to 40° C. or below, slightly cooled, andin some instances may be frozen. In some embodiments, a vaccinecomposition of the present disclosure may be made without freeze dryingor spray drying. The final formulation may then be cast onto a flatbacking surface in a laminar flow hood and allowed to form an amorphoussolid at ambient temperatures (15-20° C.). Examples of suitable backingsurfaces may include, but are not limited to, thin layers of aluminum,Teflon, silicate, polyetheretherketone, low density polyethylene, ethylcellulose, etc. Once the process is complete the vaccine composition canbe peeled from the backing and placed in the mouth for immunizationpurposes and/or stored at ambient temperature for up to three years frommanufacture.

In another embodiment, a vaccine composition of the present disclosuremay be made by contacting an amorphous solid with an antigen, oroptionally, mixing an antigen with one or more excipients (surfactants,sugars, starches, etc.) and contacting the amorphous solid with themixture so as to dispose the antigen within the amorphous solid. In someembodiments, the mixture is then allowed to dry, which is then ready foradministration.

In some embodiments, vaccine compositions of the present disclosure mayfurther comprise a protective layer disposed on a surface of anamorphous solid comprising an antigen. Exemplary protective layers mayinclude, but are not limited to, an additional layer(s) of film, such aspolyethylene, polyurethane, polyether etherketone, etc., and/or anadditional layer(s) of an amorphous solid that does not contain anyantigen.

The amount of antigen that may be used in a vaccine composition of thepresent disclosure may vary greatly depending upon the type of antigenused, the formulation used to prepare the vaccine composition, the sizeof the amorphous solid, the solubility of the antigen, etc. One ofordinary skill in the art with the benefit of this disclosure will beable to determine a suitable amount of antigen to include in a vaccinecomposition of the present disclosure. In one embodiment, a vaccinecomposition may comprise about 1×10⁶ to about 1×10¹³ virus particles fora virus-based vaccine or about 1×10³ to about 1×10¹³ colony formingunits for a bacteria-based vaccine or about 0.1 mg-1 g of protein forsubunit vaccines.

It is also important to note that when formulating a vaccine compositionof the present disclosure one must also consider any toxicity and/oradverse effects. Furthermore, in an effort to create a stable vaccinecomposition, it may also be important to identify a ratio of ingredientsthat interacts with water and the antigen in a manner that preventscrystallization during drying Formation of water crystals will puncturethe virus coat or bacterial wall and compromise the overall potency ofthe vaccine. Formulations that do this to the highest degree are said toform glasses.

Any substantially solid surface can be used for casting and/or dryingcompositions of the embodiments. For example, the surface can be apolymer (e.g., plastic) or a metal surface. In some aspects, the surfacehas a low coefficient of friction (e.g., a siliconized, non-sticksurface) to provide easy removal of films. In some embodiments, a glassplate can be used for casting of the vaccine compositions. Compositionthat have been cast on a surface can then be dried, for instance, undera controlled, laminar flow of air at room temperature, or underrefrigerated conditions. Similarly, vaccine compositions suitable foruse in the present disclosure can be prepared in a single-layer ormulti-layers.

In general, the vaccine compositions of the present disclosure may beformulated so as to dissolve in a relatively short period of time, forexample, from about 5 to 60 seconds or 1 to 30 minutes. Whenadministered, a vaccine composition of the present disclosure may behandled by a portion of the composition that does not contain an antigenand may be placed in the upper pouch of the cheek for buccal delivery,or far under the tongue for sublingual delivery or reconstituted andutilized as a solution for inhalation or as a nasal spray.

In some embodiments, the compositions and methods of the presentdisclosure may also be used as a means for treating a variety ofmalignant cancers. For example, the vaccine compositions of the presentdisclosure can be used to mount both humoral and cell-mediated immuneresponses to particular proteins specific to the cancer in question,such as an activated oncogene, a fetal antigen, or an activation marker.Such tumor antigens include any of the various MAGEs (melanomaassociated antigen E), including MAGE 1, 2, 3, 4, etc, any of thevarious tyrosinases; MART 1 (melanoma antigen recognized by T cells),mutant ras; mutant p53; p97 melanoma antigen; CEA (carcinoembryonicantigen), among others.

In certain aspects, methods are provided for producing immunogeniccompositions in substantially solid carriers. Such a method may compriseobtaining or formulating a solution comprising sufficient stabilizers(e.g., sugars and sugar derivatives, polymers) and permeabilityenhancers (e.g., surfactants, such as a zwitterionic surfactant of theembodiments) in a solvent system (e.g., distilled deionized water,ethanol, methanol). In some cases, formulation is such that the totalamount of solid components added to the solvent are within theconcentration of 10%-90% w/w. This suspension can be prepared bystirring, homogenization, mixing and/or blending these compounds withthe solvent. In some cases, small portions of each component (˜ 1/10 thetotal amount) are added to the solvent and the solution mixed beforeadding additional portions of the same agent or a new agent.

In certain aspects, once each stabilizer and permeability enhancer isadded, the bulk solution is placed at 4° C. for a period of time between2-24 hours. In some aspects, the bulk solution is subjected additionalhomogenization, such as sonication (e.g., for a period of 5-60 minutes)to remove trapped air bubbles in the preparation. After sonication iscomplete, the antigen, such as a viral vector, is added to thepreparation. In some cases, the amount of antigen will range from of0.1-30% of the total solid concentration. Adjuvants, optionally, canalso be added at this time. In some aspects, the amount of adjuvantcompounds will range from 0.005-10% of the total solid concentration.Again, in some aspects, these agents will be added by gentle stirring(e.g., 10-50 rpm) so as to not induce airpockets/bubble formation in thefinal preparation.

In some cases, the preparation is then slowly piped into molds of ashape suitable for the application. The molds can be constructed of avariety of materials including, but not limited to, stainless steel,glass, silicone, polystyrene, polypropylene and other pharmaceuticalgrade plastics. In some cases, the preparation can be placed in themolds by slowly pouring by hand or by pushing the preparation through anarrow opening on a collective container at a slow controlled rate(e.g., 0.25 ml/min) to prevent early hardening and/or bubble formationin the final film product. In certain preferred aspects, films will bepoured to a thickness of 12.5-1000 μm. In some aspects, molds forcasting of films will be sterilized by autoclaving and placed in laminarair flow hoods prior to casting.

In further aspects, molds may also be lined with a peelable backingmaterial suitable for protection of the film product. Suitable backingsinclude, without limitation, aluminum, gelatin, polyesters,polyethylene, polyvinyl and poly lactic co-glycolide polymers, wax paperand/or any other pharmaceutically acceptable plastic polymer.

In some cases, cast films will remain at ambient temperature (e.g.,20-25° C.), such as in a laminar flow hood for 2-24 hours after whichtime a thin, peelable film will be formed. In some cases, this film maybe opaque or translucent. In some cases, films are stored at roomtemperature under controlled humidity conditions. However, in certainaspects, films can be stored at lower temperatures, such as at 4° C.,under controlled humidity as well.

In certain aspects, multilayer films can also be created at this time byapplying a second coating of as solution containing the same antigen asthe first layer or another different adjuvant/antigen system to the thinfilm. Again, in some cases, this will remain at ambient temperature(e.g., 20-25° C.), such as in a laminar flow hood, for an additional2-24 hours after which time a thin, peelable film will be formed. Againthe film may be opaque or translucent.

In certain cases, films will be dissolved in a solution prior to use.For example, water or warmed saline (e.g., ˜37° C., body temperature)may be used. In some cases, the resulting solution can be screened forantigen confirmation and activity to determine the effectiveness of theformulation to retain the potency of the preparation over time.

To facilitate a better understanding of the present invention, thefollowing examples of certain aspects of some embodiments are given. Inno way should the following examples be read to limit, or define, theentire scope of the invention.

Example 1 Materials and Methods (Examples 2-8)

Materials—

Acepromazine was purchased from Fort Dodge Laboratories (Atlanta, Ga.).Ketamine was purchased from Wyeth, Fort Dodge, Animal Health, (OverlandPark, Kans.). Dulbecco's phosphate-buffered saline (DPBS), xylazine,tresyl chloride activated monomethoxypoly(ethylene)glycol, L-lysine,Poly(ethylene) glycol 3000, ethyl acetate, poly(lactide-co-glycolide)copolymers (PLGA, 50:50 lactide:glycolide), polyvinyl alcohol (PVA),glutaraldehyde (Grade I, 25% in water), o-phenylenediamine, sucrose (USPgrade), D-mannitol (USP grade), D-sorbitol (USP grade), bovine serumalbumin (RIA grade), Brefeldin A, potassium ferricyanide and potassiumferrocyanide were purchased from Sigma-Aldrich (St. Louis, Mo.). Eosin Yand Tween 20 were purchased from Fisher Scientific (Kalamazoo, Mich. andPittsburgh, Pa. respectively). Melezitose monohydride was purchased fromMP Biomedicals (Solon, Ohio) and raffinose pentahydrate from Alfa Aesar(Ward Hill, Mass.). Sodium hydroxide, potassium phosphate monobasic,potassium phosphate dibasic and sodium dodecyl sulfate were purchasedfrom Mallinckrodt Baker (Phillipsburg, N.J.). Pluronic F68 was purchasedfrom BASF (Mount Olive, N.J.). Dulbecco's Modified Eagle's medium(DMEM), RPMI-1640, Minimal Essential Medium (MEM) and L-glutamine werepurchased from Mediatech (Manassas, Va.). Fetal bovine serum (qualified,US origin), penicillin and streptomycin were purchased from Gibco LifeTechnologies (Grand Island, N.Y.). Sodium pyruvate and non-essentialamino acids were purchased from Lonza (Walkersville, Md.).5-bromo-4-chloro-3-indolyl-3-D-galactoside (X-gal) was purchased fromGold Biotechnology (St. Louis, Mo.). N-dodecyl-β-D-maltopyranoside(nDMPS), poly (Maleic Anhydride-alt-1-Decene) substituted with3-(Dimethylamino) Propylamine (PMAL C8, formula weight (F.W.) 8,500),poly (Maleic Anhydride-alt-1-Tetradecene) substituted with3-(Dimethylamino) Propylamine (PMAL C12, F.W. 12,000) and poly (MaleicAnhydride-Alt-1-Octadecene) substituted with 3-(Dimethylamino)Propylamine (PMAL C16, F.W. 39,000) were purchased from Anatrace(Maumee, Ohio). The TELRTFSI (SEQ ID NO: 1) peptide was purchased fromNew England Peptide (Gardner, Mass.). The negative control peptide(YPYDVPDYA (SEQ ID NO: 2)) was purchased from GenScript (Piscataway,N.J.). Antibodies used for ELISPOT and flow cytometry, Cytofix/Cytopermand Perm/Wash reagents were purchased from BD Pharmingen (San Diego,Calif.).

Adenovirus Production—

Four different recombinant adenoviruses were used in these examples. Allwere first generation E1/E3 deleted recombinant adenovirus serotype 5vectors that differed only by transgene expression cassettes. Twovectors, AdlacZ, expressing E. coli beta-galactosidase and AdGFP,expressing green fluorescent protein were used for rapid screening ofthe transduction efficiency of formulations in vitro and in vivo due tothe ease by which their transgene products could be visualized andquantitated. AdNull, an E1/E3 deleted adenovirus 5 vector with a similargenetic backbone as the other viruses used in these examples except thatit does not contain a marker transgene expression cassette, was used toinduce pre-existing immunity to adenovirus 5 in mice prior toimmunization. These viruses were each amplified in HEK 293 cells (ATCCCRL-1573 Manassas, Va.). They were purified from cell lysates by bandingtwice on cesium chloride gradients and desalted over Econo-Pac 10 DGdisposable chromatography columns (BioRad, Hercules, Calif.)equilibrated with potassium phosphate buffer (KPBS, pH 7.4). Theconcentration of each preparation was determined by UVspectrophotometric analysis at 260 nm and by an infectious titer assayas described¹⁸. Preparations with a ratio of infectious to physicalparticles of 1:100 were used for these examples. For immunization andchallenge studies, an E1/E3 deleted recombinant adenovirus serotype 5vector expressing a codon optimized full-length Ebola glycoproteinsequence under the control of the chicken β-actin promoter(Ad-CAGoptZGP) was amplified in HEK 293 cells and purified as describe d(Choi et al., 2013). Concentration of this and AdNull was determined byUV spectrophotometric analysis at 260 nm and with the Adeno-X RapidTiter Kit (Clontech, Mountain View, Calif.) according to themanufacturer's instructions. Preparations with infectious to physicalparticle ratios of 1:200 of each of these viruses were used in theseexamples.

PEGylation of Adenovirus—

PEGylation was performed according to established protocols (Croyle etal., 2000; Wonganan et al., 2010). Characterization of thesepreparations revealed significant changes in the biophysical propertiesof the virus such as the PEG-dextran partition coefficient and peakelution times during capillary electrophoresis (Wonganan et al., 2010).Approximately 18,245±546 PEG molecules were associated with each virusparticle in the examples outlined here as determined by a PEG-biotinassay (Croyle et al., 2005).

PLGA Microspheres—

PLGA microspheres were prepared using a standard water-in-oil-in-water(W/O/W) double emulsion and solvent evaporation method (Danhier et al.,2012). One milliliter of virus (5×10¹² virus particles) was added toethyl acetate containing 100 mg PLGA. The primary water-in-oil emulsionwas prepared by homogenization for 30 seconds and was then added to 10milliliters of an aqueous solution containing 5% (w/v) PVA. Thesecondary W/O/W emulsion was prepared by homogenization for 60 secondsand further agitated with a magnetic stirring rod for 2 hours at 4° C.to evaporate the co-solvent. Microspheres were collected bycentrifugation at 2,000 rpm for 3 minutes and washed five times withsterile KPBS. The diameter of the microspheres fell between 0.3 to 5 μmwith an average particle size of 2.06±1.4 μm as determined by dynamiclight scattering using a DynaPro LSR laser light scattering device anddetection system (Wyatt Technology, Santa Barbara, Calif.).Regularization histograms and assignment of hydrodynamic radii values tovarious subpopulations within the sample were calculated using DynaLSsoftware (Wyatt). The amount of virus embedded in the microspheres wasdetermined by digesting a portion of each preparation with 1 N NaOH for24 hours. The average encapsulation efficiency of this process was21.6±4.4% (n=6). Aliquots of each preparation were dried, placed insterile, light resistant containers and stored at room temperature forevaluation of stability over time. Release profiles of each preparationwere determined by placing 10 mg of microspheres in 0.5 ml sterile KPBSon a magnetic stir plate (Corning, Tewksbury Mass.) in a 37° C.incubator. Each day, microspheres were collected by centrifugation, thesupernatant collected and replaced with KPBS pre-warmed to 37° C. Thenumber of infectious virus particles released from microspheres wasdetermined by serial dilution of collected samples and subsequentinfection of Calu-3 cells (ATCC, HTB-55), an established model of theairway epithelia (Ong et al., 2013).

Example 2 In Vitro Screening of Formulations

Two vectors, AdlacZ, expressing E. coli beta-galactosidase and AdGFP,expressing green fluorescent protein were used for rapid screening ofthe transduction efficiency of formulations due to the ease by whichtheir transgene products could be visualized and quantitated.Formulations were prepared at five times the working concentration,sterilized by filtration and diluted with freshly purified virus in KPBS(pH 7.4) prior to use. Two hundred microliters of formulation containingvirus (MOI 100) in the absence or presence of anti-adenovirus antibodieswere added to differentiated Calu-3 cells seeded at a density of 1.25×10cells/well in 12 well plates. Formulations remained in contact with cellmonolayers for 2 hours at 37° C. in 5% CO₂. Cytotoxicity was assessed bymeasuring lactate dehydrogenase (LDH) release into the formulation witha standard cytotoxicity kit (Roche Applied Science, Indianapolis, Ind.)according to the manufacturer's instructions. Complete lysis wasachieved by adding 1% sodium dodecyl sulfate to cells not exposed toformulations (positive control). Transduction efficiency was measured 48hours later by either histochemical staining, visual inspection andquantitation of cells expressing beta-galactosidase or by flow cytometryto quantitate cells expressing GFP.

Example 3 Mouse Studies

All procedures were approved by the Institutional Animal Care and UseCommittees at The University of Texas at Austin and the University ofTexas Medical Branch in Galveston and are in accordance with theguidelines established by the National Institutes of Health for thehumane treatment of animals. Two different strains of mice were used inthese examples. Male B10.Br mice (MHC H-2^(k)) were used to characterizethe immune response to Ebola glycoprotein after immunization with theAd-CAGoptZGP vector as described previously (Croyle et al., 2008; Choiet al., 2012; Choi et al., 2013). Because this strain is difficult tobreed (Lerner et al., 1992), and is often not readily available inquantities sufficient from the supplier to perform the studies outlinedin this manuscript, male C56/BL6 (MHC H-2^(d)) mice were used forinitial screening of formulations that improved transgene expression invitro with minimal cytotoxic effects. Both strains were obtained fromthe Jackson Laboratory (age 4-6 weeks, Bar Harbor, Me.).

Nasal Administration of Virus/Immunization—

Animals were housed in a temperature-controlled, 12 hour light-cycledfacility at the Animal Research Center of The University of Texas atAustin with free access to standard rodent chow (Harlan Teklad,Indianapolis, Ind.) and tap water. Animals were anesthetized by a singleintra-peritoneal injection of a 3.9:1 mixture of ketamine (100 mg/ml)and xylazine (100 mg/ml). Once deep plane anesthesia was achieved,animals were placed on their stomach. The sedate animal's head wasrested upon an empty tuberculin syringe to keep the head in an uprightposition and to minimize choking or accidental swallowing of vaccine(FIG. 9). Each mouse received a dose of 1×10 infectious particles ofunformulated or formulated vaccine by direct application in the nasalcavity. The inhalation pressure from the animal's natural breathing wassufficient to allow small droplets from the standard micropipette(Gilson, Middleton, Wis.) to gently enter the nasal cavity without theneed to forcefully inject the solution. The right nostril received 10 μLand was allowed to dry for up to 5 minutes before adding an additional10 μL to the left nostril, for a total volume of 20 μL per animal. Theanimal was observed in the relaxed position for an additional 10 minutesto guarantee comfortable breathing and ensure that the vaccine was notlost via sneezing (a rare occurrence that can result from touching theanimal's nose with the micropipette tip instead of allowing the tinydroplet to be gently pulled into the nose through natural inhalationpressure).

Establishment of Pre-Existing Immunity to Adenovirus—

A first generation adenovirus that that does not contain a transgenecassette (AdNull) was used to establish pre-existing immunity toadenovirus serotype 5 (Callahan et al., 2006). Twenty-eight days priorto vaccination, mucosal PEI was induced by placing 5×10¹⁰ particles ofAdNull in the nasal cavity as described above under the immunizationprotocol. Twenty-four days later, blood was collected via the saphenousvein and serum screened for anti-Ad neutralizing antibodies (NABs) asdescribed below. At the time of vaccination, animals had an averageanti-Ad circulating NAB titer of 315±112 reciprocal dilution, whichfalls within the range of average values reported in humans afternatural infection (Barouch et al., 2011).

Challenge with Mouse-Adapted Ebola Virus—

Challenge experiments were performed under biosafety level 4 (BSL-4)conditions in an AAALAC accredited animal facility at the Robert E.Shope BSL-4 Laboratory at the University of Texas Medical Branch inGalveston, Tex. Twenty-one days post-immunization, vaccinated mice weretransported to the BSL-4 lab where they were challenged on day 28 byintraperitoneal injection with 1,000 pfu of mouse-adapted (30,000×LD₅₀)Ebola (Bray et al., 1999). After challenge, animals were monitored forclinical signs of disease and weighed daily for 14 days. Serum alanineaminotransferase (ALT) and aspartate aminotransferase (AST) levels weredetermined using AST/SGOT and ALT/SGPT DT slides on a Vitros DTSCautoanalyzer (Ortho Clinical Diagnostics, Rochester, N.Y.).

Example 4 In Vitro Studies with Immunized Samples

ELISPOT—

ELISpot assays were performed using the ELISpot Mouse Set (BDPharmingen) according to the manufacturer's instructions. Mononuclearcells were isolated from the spleen and bronchoalveolar lavage fluid asdescribed previously (Choi et al., 2013), washed twice with completeDMEM and added to wells of a 96-well ELISpot plate (5×10⁵ cells/well)with the TELRTFSI peptide ((SEQ ID NO: 1), 0.5 μg/well) that carries theEbola virus glycoprotein immunodominant MHC class I epitope for micewith the H-2^(k) haplotype (B10.Br) (Rao et al., 1999). Negative controlcells were stimulated with an irrelevant peptide, which carries abinding sequence for influenza hemagglutinin (YPYDVPDYA (SEQ ID NO: 2),0.5 μg/well). Spots were counted using an automated ELISpot reader(CTL-ImmunoSpot® S5 Micro Analyzer, Cellular Technology Ltd., ShakerHeights, Ohio).

Multi-Parameter Flow Cytometry—

Splenocytes (2×10⁶) isolated from immunized mice were cultured withTELRTFSI peptide ((SEQ ID NO: 1), 0.5 μg/well) and 1 μg/ml Brefeldin Afor 5 hours at 37° C. in 5% CO₂. Negative control cells were incubatedwith the YPYDVPDYA peptide ((SEQ ID NO: 2), 0.5 μg/well). Followingstimulation, cells were surface stained with anti-mouse CD8a antibodies(1:150 in DPBS) and followed by intracellular staining with anti-mouseIFN-γ, TNF-α and IL-2 antibodies as described (Choi et al., 2013).Positive cells were counted using four-color flow cytometry (FACSFortessa, BD Biosciences, Palo Alto, Calif.). Over 500,000 events werecaptured per sample. Data were analyzed using FlowJo software (TreeStar, Inc., Ashland, Oreg.).

CFSE Assay—

Splenocytes were isolated 42 days post-vaccination, stained using theVybrant CFDA SE Cell Tracer kit (Invitrogen, Carlsbad, Calif.), seededat a concentration of 5×10⁵ cells/well in 96 well plates and culturedfor 5 days at 37° C. with 5% CO₂ in the presence of the TELRTFSI (SEQ IDNO: 1) or YPYDVPDYA (SEQ ID NO: 2) peptides (0.5 μg/well) as describedpreviously (Choi et al., 2012). Cells were incubated with a cocktail ofantibodies (perCPCy5.5 labeled-anti-mouse-CD8, PElabeled-anti-mouse-CD44, and allophycocyanin (APC)labeled-anti-mouse-CD62L, 1:150) and analyzed by flow cytometry withover 1,000,000 events captured per sample.

Characterization of Ebola Glycoprotein-Specific Antibodies—

Flat bottom, Immulon 2HB plates (Fisher Scientific, Pittsburgh, Pa.)were coated with purified Ebola virus GP₃₃₋₆₃₇ ΔTM-HA (3 μg/well) in PBS(pH 7.4) overnight at 4° C. (Lee et al., 2009). Heat-inactivated serumsamples were diluted (1:20) in PBS. One hundred microliters of eachdilution were added to antigen-coated plates for 2 hours at roomtemperature. Plates were washed 4 times and incubated withHRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b and IgM (1:2,000,Southern Biotechnology Associates, Birmingham, Ala.) antibodies inseparate wells for 1 hour at room temperature. Plates were washed andsubstrate solution added to each well. Optical densities were read at450 nm on a microplate reader (Tecan USA, Research Triangle Park, N.C.).

Adenovirus Neutralizing Assay—

Heat-inactivated serum was diluted in twofold increments starting from a1:20 dilution. Each dilution was incubated with AdlacZ (1×10⁶ pfu) for 1hour at 37° C. and applied to HeLa cells (ATCC # CCL-2) seeded in96-well plates (1×10⁴ cells/well). After this time, 100 μl of DMEMsupplemented with 20% FBS were added to each well. Twenty-four hourslater, beta-galactosidase expression was measured by histochemicalstaining. Dilutions that reduced transgene expression by 50% werecalculated using the method of Reed and Muench (reed et al., 1938). Theabsence of neutralization in samples containing medium only (negativecontrol) and FBS (serum control) and an average titer of 1:1,280±210read from an internal positive control stock serum were the criteria forqualification of each assay.

Statistical Analysis—

Data were analyzed for statistical significance using SigmaStat (SystatSoftware Inc., San Jose, Calif.) by performing a one-way analysis ofvariance (ANOVA) between control and experimental groups, followed by aBonferoni/Dunn post-hoc test when appropriate. Differences in the rawvalues among treatment groups were considered statistically significantwhen p<0.05.

Example 5 Characterization of Formulations

A variety of novel formulations were identified, prepared and evaluatedfor their ability to maintain or improve transduction efficiency of theadenovirus with minimal cytotoxicity in Calu-3 cells, an in vitro modelof the human respiratory epithelium (Ong et al., 2013). Over 400formulations were assessed using a recombinant adenovirus expressing E.coli beta-galactosidase that differed from the inventors' vaccineconstruct only by the transgene cassette. This virus was chosen forthese studies because it was available in sufficient quantity to supportthe high-throughput screening approach used by the inventors and for theease by which the beta-galactosidase transgene product could bevisualized and quantitated in vitro and in vivo. Data summarized hereillustrate the inventors' heuristic approach where the number offormulation candidates tested in vitro is significantly reduced prior tothe first in vivo screen for transduction efficiency and safety andfurther reduced to a select few for characterization of the immuneresponse and subsequent evaluation of protection from lethal exposure torodent-adapted Ebola.

In Vitro Characterization of Formulated Adenovirus Preparations—

While many of the formulations included in the initial screen couldmaintain the stability of the adenovirus at ambient temperatures (datanot shown), very few improved the in vitro transduction efficiency ofthe virus above that seen from virus formulated in saline. However, apreparation of 5% w/v sucrose increased transduction efficiency by afactor of 1.96 with respect to virus formulated in saline (pH 7.4, FIG.21A). Pluronic F68 (0.005% w/v), mannitol (1.25% w/v) and melezitose(1.25% w/v) alone each increased transduction by a factor of 1.78, 1.61and 1.51, respectively. Formulations consisting of either raffinose orsorbitol alone at a 1.25% (w/v) concentration did not significantlyenhance transduction (p=0.06). A multi-component formulation, F3,consisting of sucrose (10 mg/ml), mannitol (40 mg/ml) and 1% v/vpoly(ethylene) glycol 3,000, previously found to stabilize the virus atambient temperatures for extended periods of time (Renteria et al.,2010), increased transduction fivefold (p=0.03). A formulation of 100 μMnDMPS was the second most efficacious formulation, however, significantcytoxicity (>80% cell lysis) was observed in cultures treated with thisformulation. Less than 1% lysis was observed in cells treated with F3,making it suitable for further evaluation in vivo (data not shown).

In an effort to improve transduction efficiency in the presence ofanti-adenovirus neutralizing antibodies, a method for encapsulation ofadenovirus with the bio-compatible polymer poly(lactic-co-glycolic acid)was also developed (Choi et al., 2012). Only preparations that werecapable of releasing active virus at concentrations relevant forimmunization after storage at ambient temperatures would be consideredfor further in vivo testing. Approximately 10±0.43% of the virusparticles embedded in the polymer matrix were rendered uninfectiousduring processing, leaving the average virus concentration to be1.08±0.5×10⁹ infectious virus particles (ivp) per milligram ofmicrospheres. Infectious virus particles were released for 14 days afterwhich virus could no longer be detected (FIG. 1B). Approximately 50% ofthe total amount of infectious virus embedded in freshly made polymerbeads and in beads stored at 25° C. for 7 days was released within 24hours (FIG. 1C). Storage of the beads at room temperature for 7 days didnot significantly impact release rate as 1.34×10⁸ ivp were released perday from freshly prepared beads and 1.76×10⁸ ivp released per day afterthis time. Storage at room temperature for one month increased the rateof release to 3.5×10⁸ ivp/day and promoted release of the entire dose(97.95%) within 48 hours (FIG. 1C).

In Vivo Transduction Efficiency of Formulated Adenovirus Preparations inNaïve Mice and Those with Pre-Existing Immunity—

Based upon their ability to improve transduction efficiency and maintainvirus stability, the F3 formulation and PLGA microspheres were selectedfor further evaluation of their effect on the transduction efficiency ofadenovirus in naïve mice and those with pre-existing immunity (PEI) toadenovirus. PEGylated virus was also selected for evaluation as avaccine platform since the inventors have previously shown that covalentattachment of poly(ethylene) glycol to the virus capsid improves thetransduction efficiency in animals that have been exposed to adenovirus(Croyle et al., 2001). Intranasal administration of AdlacZ, the modelvirus used for in vitro screening studies summarized in FIGS. 1A-C, ineach respective formulation resulted in high levels of transgeneexpression in epithelial cells of the conducting airways of naïve mice 4days after treatment (FIGS. 2A-2D). More importantly, each formulationsignificantly improved transgene expression in mice with pre-existingimmunity to adenovirus (FIGS. 2F-2H) with respect to unformulated virus(FIG. 2E).

Example 6 Characterization of Immune Response

The T Cell Response: Magnitude—

Since the transduction efficiency data in mice with pre-existingimmunity to adenovirus looked promising for each formulation, the immuneresponse elicited by each formulation was evaluated in B10.Br mice withthe Ad-CAGoptZGP vector as described previously (Croyle et al., 2008;Choi et al., 2012; Choi et al., 2013). The systemic antigen-specific Tcell response generated by each formulation candidate was evaluated byquantitation of IFN-γ secreting mononuclear cells (MNCs) in the spleenby ELISpot. There was no significant difference in the amount ofantigen-specific cells present in samples obtained from naïve animalsimmunized with PEGylated, PLGA encapsulated or unformulated virus(p>0.05, FIG. 3A). Samples from mice immunized with the F3 formulationcontained slightly more antigen-specific cells than those from micegiven unformulated vaccine (486.7±4.8 spot-forming cells (SFCs)/millionMNCs, F3 vs. 414.7±27.6 SFCs/million MNCs, Unform.). In contrast to whatwas observed in naïve animals, PEI significantly decreased the number ofactivated IFN-γ secreting MNCs in the spleens of animals given eachpreparation except in those given the PEGylated vaccine (317.3±58.2spot-forming cells (SFCs)/million mononuclear cells (MNCs), naïve vs.234.7±54.3 SFCs/million MNCs, PEI, FIG. 3A). The most significantreduction in IFN-γ secreting MNCs was observed in animals given themicrosphere preparation (426.7±33.8 SFCs/million MNCs, Naive vs.57.3±7.1 SFCs/million MNCs, PEI). Pre-existing immunity alsosignificantly reduced the number of IFN-γ secreting cells recovered frombronchioalveolar lavage (BAL) fluid in mice given unformulated vaccine(1,513.3±63.6 SFCs/million MNCs, naïve vs. 526.7±98.2 SFCs/million MNCs,PEI, p<0.01, FIG. 3B). This response was not compromised in animalsgiven the PEGylated and PLGA encapsulated vaccines (PEG: 266.7±54.6SFCs/million MNCs, naïve vs. 580±61.1 SFCs/million MNCs, PEI; PLGA:1280±90.2 SFCs/million MNCs, naïve vs. 1360±231.8 SFCs/million MNCs,PEI).

The T Cell Response: Quality—

Both the quantity and quality of antigen-specific CD8⁺ T cells inducedby a vaccine platform significantly contribute to protection from avariety of infectious diseases such as AIDS, malaria, and hepatitis C(Fraser et al., 2013). In animal models of infection, the quality of theantigen-specific T cell response can be assessed by stimulation ofsplenocytes, intracellular staining and multi-parameter flow cytometryto characterize the diversity of CD8⁺ T cell populations induced afterimmunization (Zielinski et al., 2011). The presence of poly-functionalCD8⁺ T cells, capable of producing several cytokines (IFN-γ, IL-2, andTNF-α) in response to the antigen, has been found to correlate with areduction in circulating antigen and viral load since they are known tobe the most responsive cells early in the infection process (Seder etal., 2008). Thus, strategies to increase the presence of cells capableof producing variety of cytokines and chemokines in response to apathogen are part of many immunization strategies (Sallusto et al.,2010; Coffman et al., 2010). In this context, functional analysis ofcytokine producing CD8⁺ T cells at the single-cell level was performedto determine the ability of the formulations described herein to improvethe quality of the antigen-specific CD8⁺ T cell response. As part ofthis analysis, the inventors were able to delineate seven distinctcytokine-producing cell populations based upon IFN-γ, IL-2, and TNF-αsecretion patterns.

As stated above, the relative frequency of cells that produce all threecytokines defines the quality of the vaccine-induced CD8⁺ T cellresponse. In naïve mice, each formulation increased the number ofpoly-functional CD8⁺ T cells, with the PLGA encapsulated vaccineproducing the highest amount of these cells (IFN-γ⁺IL-2⁺TNF-α⁺,37.1±5.04%, FIG. 3C). Prior exposure to adenovirus in the nasal mucosareduced the quality of the response generated by the unformulatedvaccine (23.9±3.24%, Naive vs. 19.1±6.76%, PEI) and F3 (27.2±4.60%,Naive vs. 14.8±2.85° %, PEI) while the response induced by the PEGylatedvaccine was not compromised (24.8±3.69%, Naive vs. 26.9±4.74%, PEI, FIG.3D). The poly-functional response was somewhat strengthened in mice withprior exposure to adenovirus given the PLGA microspheres (37.1±5.04%,Naive vs. 41.7±7.88%, PEI). This effect was also seen 42 days afterimmunization.

The T Cell Response: Memory—

Antigen-specific CD8⁺ memory T cells are crucial components of long-termprotection against viral infections. In order to predict the long-termefficacy of the formulated vaccines of the invention, the inventorsevaluated the effector memory CD8⁺ T cell response with a CFSEproliferation assay. Forty-two days after immunization, splenocytesisolated from naive mice given the unformulated vaccine contained8.8±1.02% effector memory CD8⁺ T cells capable of proliferating inresponse to an Ebola virus glycoprotein-specific MHC I-restrictedpeptide (FIG. 3E). The number of effector memory CD8⁺ T cells was lowerin samples harvested from animals immunized with the other formulations.Prior exposure to adenovirus significantly reduced the memory responsein mice given the F3 formulation, PEGylated and unformulated vaccine.The response elicited by PLGA encapsulated vaccine was suppressed bypre-existing immunity to a lesser degree than that observed in the othertreatment groups (2.32±0.09% vs. 0.85±0.08, F3 vs. 0.72±0.04, PEG).

The Anti-Ebola Virus Antibody Response—

The inventors have previously found that prior exposure to adenovirussignificantly reduced antibody-mediated immune response to Ebolaglycoprotein in mice and guinea pigs (Choi et al., 2013). Morespecifically, the inventors also found that a reduction inglycoprotein-specific IgG1 antibodies correlated with poor survivalafter challenge with rodent-adapted Ebola. Thus, the inventors evaluatedtotal anti-Ebola glycoprotein-specific immunoglobulin (IgG) and IgGisotypes in serum to determine if each formulation could counterbalancethe effect of prior mucosal exposure to adenovirus on B cell-mediatedimmune responses (FIGS. 4A-4B). Each formulation significantly increasedthe amount of each antibody isotype specific for Ebola glycoprotein (GP)in naïve mice (FIG. 4A). IgG2a levels in mice given PLGA microsphereswas the only deviation from this trend as it was reduced by 29.9% withrespect to unformulated virus (FIG. 4A). Prior exposure to adenovirussignificantly reduced each anti-Ebola GP-specific IgG isotype evaluated(FIG. 4B). Although IgG2b levels in samples collected from miceimmunized with the F3 formulation doubled, IgG1 and IgG2a levels werenot significantly different from that seen in animals given unformulatedvirus. IgG1 and IgG2b levels in mice immunized with PLGA microsphereswere 9.5 and 1.3 times that found in samples from mice givenunformulated vaccine (FIG. 4B). PEI to adenovirus reduced IgG2b levelsby 45.9% in mice given PEGylated vaccine. IgG1 and IgG2a could not bedetected in serum of mice immunized with this preparation. Trace levelsof Ebola GP-specific IgM antibodies were found in serum from mice giventhe PLGA and PEGylated preparations.

Survival From Lethal Challenge—

A marked reduction in the quality of the T cell response and in Th2 typeantibody responses were found to be indicative of poor protectionagainst lethal infection with Ebola virus in animals with PEI toadenovirus (Choi et al., 2013). Using this criteria, the inventorsdecided that mice immunized with vaccine in formulation F3 would not besubject to challenge with a lethal dose of a mouse-adapted variant ofEbola (MA-EBOV) since neither facet of the immune response was notablyimproved by the formulation in mice with PEI to adenovirus. All of thenaïve mice given unformulated vaccine and the PLGA microspherepreparation survived lethal challenge with MA-EBOV (1,000pfu≃30,000×LD₅₀, FIG. 5A). Twenty five percent of naïve mice given thePEGylated vaccine succumbed to infection. Sixty percent of the animalswith PEI to adenovirus that were immunized with unformulated vaccinesurvived challenge. Eighty percent of mice with prior exposure toadenovirus that were immunized with the PEGylated preparation did notsurvive challenge. This group also demonstrated the most notable drop inbody weight during the course of infection (FIG. 5B). Samples taken fromthis group also revealed sharp elevations in ALT (842±342 U/L) and AST(602±298 U/L), indicative of severe liver damage from infection (FIG.5C). The PLGA microsphere preparation protected 80% of the mice with PEIto adenovirus from challenge. Serum ALT (195±7.25 U/L) and AST (232±10.1U/L) levels were significantly lower in this treatment group withrespect to those from animals given only saline (ALT 1,913.6±228.6 U/L;AST 2,152±394.77 U/L) for which the challenge was uniformly lethal andfrom mice with PEI given unformulated vaccine (ALT: 879±197 U/L; AST:898±241 U/L, p<0.01).

Example 7 An In Vitro Assay for Quantitative Evaluation of TransductionEfficiency of Formulated Virus in the Presence of NeutralizingAntibodies

Because the PLGA and PEGylated preparations did not fully protect micewith PEI to adenovirus from lethal challenge, a secondary effort toidentify formulations to improve survival was initiated. Based upon theinitial results with the maltoside, nDMPS, the inventors sought toidentify compounds with similar properties but reduced toxicity profilesfor further testing. Evaluation of transduction efficiency in thepresence of neutralizing antibody was also included as a more stringenttest to predict in vivo performance of formulation candidates. Threedifferent amphiphols, differing only in the length of carbon chain inthe hydrophobic region of the molecule were first evaluated for theirability to preserve the transduction efficiency of the model AdlacZvector in Calu-3 cells in the presence of neutralizing antibodies.Transduction efficiency of the virus in a formulation of 10 mg/ml ofpoly (Maleic Anhydride-alt-1-Decene) substituted with 3-(Dimethylamino)Propylamine (referred to as F8) was reduced from 2.58±0.03×10⁷ to1.94±0.14×10⁷ ivp/ml when the anti-adenovirus 5 antibody concentrationin the infection media increased from 0.5 N.D.₅₀ to 5 N.D.₅₀ (FIG. 6A).Virus formulated with 10 mg/ml poly (Maleic Anhydride-alt-1-Tetradecene)substituted with 3-(Dimethylamino) Propylamine (F12) experienced themost significant drop in transduction efficiency when antibodyconcentration was increased from 0.5 N.D.₅₀ to 5 N.D.₅₀(74% reduction,1.64±0.18×10⁷ (0.5 N.D.₅₀), to 4.28±0.48×10⁶ (5 N.D.₅₀) lfu/ml).Transduction efficiency of the virus formulated with 10 mg/ml poly(Maleic Anhydride-Alt-1-Octadecene) substituted with 3-(Dimethylamino)Propylamine (F16) in the presence of 5 N.D.₅₀ neutralizing antibody wasnot significantly different from that in the presence of the 0.5 N.D.₅₀concentration (p=0.08, FIG. 6A). This compound also had a very favorabletoxicity profile as formulations of 1 and 10 mg/ml were cytotoxic toonly 1.9±0.47 and 1.8±0.61% of the Calu-3 cell population respectively(FIG. 6B). Increasing the concentration to five times that of theeffective concentration (50 mg/ml) was still well tolerated by theCalu-3 cell monolayer with 3.63±0.35% lysis noted.

Before the vaccine formulated with the F16 preparation was tested invivo, it underwent an additional round of screening to confirm thattransduction efficiency was adequately improved in the presence ofneutralizing antibody. In order to increase the sensitivity of thisassay and make it a better predictor of in vivo performance, theinventors decided to incorporate a model a recombinant adenovirus 5vector expressing green fluorescent protein (AdGFP) into the testformulations and evaluate transduction efficiency byfluorescence-activated cell sorting (FACS). In order to generate datathat was clinically relevant, the assay was the virus was incubated withfive different solutions containing a series of neutralizing antibodyconcentrations spanning those found in the general population^(28, 41).Cells infected with the virus were quantitated by flow cytometry 48hours after infection. While 8.58% of the monolayer infected withunformulated virus expressed the transgene, the F16 formulationincreased transduction efficiency to 38.8% (FIG. 6C). This assay allowedthe inventors to see significant differences in transduction efficiencyof the formulated virus in the presence of the 0.5 N.D.₅₀ and 5 N.D.₅₀antibody concentrations that was not detected by the infectious titerassay (3.44%, 0.5 N.D.₅₀, vs. 34.4%, 5 N.D.₅₀). Although the formulationimproved transduction efficiency of the virus over a wide range ofantibody concentrations, the limit of this improvement was reached atthe 50 N.D.₅₀ concentration where the formulation could no longerprotect the virus from neutralization.

Adenovirus formulated with the F16 compound was well tolerated in naïveanimals and those with PEI to adenovirus. Almost every epithelial cellin both large and small airways was transduced by virus formulated withF16 (FIG. 6D). Highly concentrated areas of transgene expression werealso found in small airways of mice with circulating neutralizingantibody levels of 262±43 reciprocal dilution given virus in thisformulation. In contrast, PEI to adenovirus prevented transgeneexpression in both large and small airways of mice given unformulatedvirus. Serum transaminases, standard indicators of adenovirustoxicity⁴², in both naïve animals and those with PEI to adenovirusimmunized with the F16 formulation were reduced by 40% with respect tosimilar treatment groups given unformulated vaccine (data not shown).

Example 8 The Immune Response Generated by Formulation F16 in Mice withPre-Existing Immunity

Because prior formulation candidates did not fully confer protection inmice in which PEI was established through the nasal mucosa, evaluationof the F16 formulation in vivo focused solely on the ability of thisformulation to improve the immune response to the encoded Ebolaglycoprotein under these specific conditions. As seen in prior studies,PEI significantly compromised the production of GP-specificIFN-γ-secreting mononuclear cells isolated from spleen (FIG. 7A) and BALfluid (FIG. 7B) in animals given unformulated vaccine (p<0.01). PEIinduced by the mucosal route also significantly reduced the frequency ofGP-specific multifunctional CD8 T cells elicited by the unformulatedvaccine (Naïve: 64.9±4.88% vs. IN PEI/Unformulated: 48.6±3.66%, p<0.05;FIG. 7C).

The F16 formulation improved the immune response in animals with PEI toadenovirus as the number of GP-specific, IFN-γ-secreting mononuclearcells isolated from the spleen of these animals was notably higher thanthat from naïve animals given unformulated vaccine (2,290±51SFCs/million MNCs, naïve vs. 2,840±110 SFCs/million MNCs, PEI/F16,p<0.05, FIG. 7A). The number of antigen-specific IFN-γ-secretingmononuclear cells isolated from the BAL fluid of animals given the F16formulation was not statistically different from that found in naïveanimals given unformulated vaccine (p=0.07, FIG. 7B). This trend wasalso observed with respect to the multifunctional CD8⁺ T cell responseas it also did not change with respect to that found in naïve animalsgiven unformulated vaccine (Naïve/unformulated: 64.9±4.88% vs. INPEI/F16 (10 mg/ml): 60.0±9.1%, p=0.055; FIG. 7C). Forty-two days afterimmunization, the effector memory CD8⁺ T cell response was alsoevaluated in mice immunized with unformulated vaccine or the F16preparation. The F16 formulation increased the memory response by afactor of 3.3 from 0.28±0.15% (unformulated) to 0.93±0.25% (F16, datanot shown). Serum from animals with pre-existing immunity to adenovirusthat were immunized with the F16 preparation contained 4 times moreanti-Ebola glycoprotein antibodies than that from animals givenunformulated vaccine (FIG. 8). Samples from these animals also contained5 times more of the IgG1 isotype and notable levels of antigen-specificIgM antibodies.

Example 9 Materials and Methods for Primate Studies (Examples 10-11)

Adenovirus Production

The E1/E3 deleted recombinant adenovirus serotype 5 vector expressing acodon optimized full-length Ebola glycoprotein sequence under thecontrol of the chicken β-actin promoter (Ad-CAGoptZGP) and a host rangemutant adenovirus serotype 5 (Ad5MUT) that can replicate in non-humanprimates were amplified in HEK 293 cells and purified as described(Richardson et al., 2009; Buge et al., 1997). Concentration of eachvirus preparation was determined by UV spectrophotometric analysis at260 nm and with the Adeno-X Rapid Titer Kit (Clontech, Mountain View,Calif.) according to the manufacturer's instructions. Preparations withinfectious to physical particle ratios of 1:37 were used in thesestudies. Buffers and reagents used in the production and purification ofeach virus preparation were of the highest quality available and weretested for the presence of endotoxin using a QCL-1000 Chromogenic LALend point assay (Cambrex Bioscience, Walkersville, Md.). All reagentscontained less than 0.1 E.U./mL, and each virus preparation containedless than 0.2 E.U./mL. Sterility of each preparation was confirmedemploying the methods outlined in the United States Pharmacopeia forparenteral products. (Sterility Tests. In the United States Pharm.,2014)

Assay for Detection of Replication Competent Adenovirus (RCA)

A two cell line bioassay was performed on each preparation to determinethe presence of RCA as described (Gilbert et al., 2014). Less than oneRCA was detected for every 3×10¹² virus particles tested.

Animal Model

Non-human primate studies were conducted under a contract at BioqualInc., Gaithersburg, Md. The animal management program of thisinstitution is accredited by the American Association for theAccreditation of Laboratory Animal Care and meets NIH standards asoutlined in the Guide for the Care and Use of Laboratory Animals. Thisinstitution also accepts as mandatory PHS policy on Humane Care ofVertebrate Animals used in testing, research, and training. Twenty malecynomolgus macaques (Macaca fascicularis) of Chinese origin were allowedto acclimate for 30 days in quarantine prior to immunization. Animalsreceived standard monkey chow, treats, vegetables, and fruits throughoutthe study. Husbandry enrichment consisted of commercial toys and visualstimulation. Two separate experiments were conducted as summarized inFIGS. 10 and 16A-16B. Specific details about the primates used in eachof these studies are summarized in Tables 1 and 2.

TABLE 1 Primate Study 1: Primate Characteristics and Treatment animal wtdose route of age no. treatment (kg) (ivp/kg) admin (years) 22457 KPBS8.05 IM 10 22473 Ad- 6.36 1.6 × 10⁸ IM 10 CAGoptZGP 40347 Ad- 6.16 1.6 ×10⁸ IM 8 CAGoptZGP 50459 Ad- 7.31 1.4 × 10⁹ IN/IT 7 CAGoptZGP 52483 Ad-6.98 1.4 × 10⁹ IN/IT 7 CAGoptZGP 52945 Ad- 6.84 1.5 × 10⁹ IN/IT 7CAGoptZGP 52165 Ad- 6.30 1.6 × 10⁹ SL 7 CAGoptZGP 62125 Ad- 5.59 1.8 ×10⁹ SL 6 CAGoptZGP 62361 Ad- 6.38 1.6 × 10⁹ SL 6 CAGoptZGP

TABLE 2 Primate Study 2: Primate Characteristics and Treatment animal wtdose route of age no. treatment (kg) (ivp/kg) admin (years) 0810091 KPBS8.7 IM 6 0805201 KPBS 6.8 IM 6 0802197 Ad- 6.2 1.6 × 10⁹  IN/IT 6CAGoptZGP 0809077 Ad- 6.5 1.5 × 10⁹  IN/IT 6 CAGoptZGP 0810003 Ad- 5.81.7 × 10⁹  IN/IT 6 CAGoptZGP 0805257 Ad- 4.9 2.0 × 10¹⁰ SL 6 CAGoptZGP0804317 Ad- 4.8 2.0 × 10¹⁰ SL 6 CAGoptZGP 0808233 Ad- 4.8 2.0 × 10¹⁰ SL6 CAGoptZGP 0809227 Ad5MUT 5.5 1.8 × 10¹⁰ IM 6 Ad- 1.8 × 10¹⁰ SLCAGoptZGP 0804819 Ad5MUT 5.2 1.9 × 10¹⁰ IM 6 Ad- 1.9 × 10¹⁰ SL CAGoptZGP0807243 Ad5MUT 4.9   2 × 10¹⁰ IM 6 Ad-   2 × 10¹⁰ SL CAGoptZGP

Primate Study 1 (Results Shown in Example 10)

The first study was conducted with 9 primates. Two animals were giventhe vaccine by intramuscular injection in a total volume of 1 mL ofpotassium phosphate buffered saline (KPBS) divided equally between theleft and right deltoid muscles. Three animals were given the vaccine bythe sublingual route by placing 50 μL of the preparation under each sideof the tongue and waiting for 15 min between doses to allow forabsorption. Three animals were given the vaccine in the respiratorytract. This was achieved by slowly dispensing two 250 μL volumes of thepreparation into each nostril and waiting for 15 min between doses toallow for absorption. The remaining dose of the vaccine (5 mL volume)was instilled into the lungs via an endotracheal tube. This route ofadministration will be referred to as respiratory immunization or asintranasal/intratracheal (IN/IT) throughout the manuscript to illustratethat the vaccine was administered to the respiratory mucosa by twodifferent routes. One primate was given 1 mL of KPBS divided equallybetween the left and right deltoid muscles. This animal was the negativecontrol. Blood was collected 6 h after immunization and on days 1, 2,and 7. Full blood chemistry panels and complete blood counts wereperformed by IDEXX BioResearch (West Sacramento, Calif.).

Primate Study 2 (Results Shown in Example 11)

A second study was conducted with 11 primates. Two animals (negativecontrols) were given 1 mL each of KPBS divided between the left andright deltoid muscles. The respiratory formulation contained sucrose (10mg/ml), mannitol (40 mg/ml) and 10 mg/mL poly(maleicanhydride-alt-1-octadecene) substituted with3-(dimethylamino)propylamine and administered as a solution to therespiratory mucosa of three animals as described for study 1. Threeanimals were given an adenovirus serotype 5 host range mutant virus toestablish pre-existing immunity (PEI) by IM injection 28 days prior toimmunization with the vaccine by the sublingual route as describedabove. Three animals with no prior exposure to adenovirus were given thevaccine by the sublingual route for comparison.

Challenge

Animals were transported to the National Microbiology Laboratory inWinnipeg and, after an acclimation period, transferred to the biosafetylevel 4 (BSL-4) laboratory there for challenge. Challenge studies wereapproved by the Canadian Science Centre for Human and Animal Health(CSCHAH) Animal Care Committee following the Guidelines of the CanadianCouncil on Animal Care. For challenge, animals were infected byintramuscular injection at two sites with a total volume of 1 mL offreshly prepared Ebola virus (strain Kikwit 95, passage 3 on VeroE6cells) of an inoculum containing 1,000 times the 50% tissue cultureinfectious dose (TCID50) in diluent (minimal essential medium containing0.3% bovine serum albumin). Ebola virus titers were confirmed (1.21×103TCID50/mL) by back-titration of the challenge preparation followingadministration of the virus. Animals were monitored daily and scored fordisease progression using an internal filovirus scoring protocolapproved by the CSCHAH Animal Care Committee. The scoring system gradedchanges from normal in the subject's posture, attitude, activity level,feces/urine output, food/water intake, weight, temperature, andrespiration and ranked disease manifestations such as a visible rash,hemorrhage, cyanosis, or flushed skin. Samples were taken for assessmentof anti-Ebola GP antibodies and full blood panels on days 3, 7, 14, 21,and 28 postchallenge and upon death. Hematological analysis of sampleswas performed in the BSL-4 lab with a Horiba ABX Scil ABC Vet AnimalBlood Counter, and blood chemistries were analyzed with a VetScan vsl(Abraxis). Surviving animals were kept until day 28.

ELISpot Assay

IFN-γ ELISpot assays were performed in triplicate according to themanufacturer's protocol (BD Biosciences, San Diego, Calif.) with 5×105peripheral blood mononuclear cells (PBMCs) per well in cRPMI media (RPMI1640, 1 mM 1-glutamine, 50 μM β-mercaptoethanol, 10% FBS and 1%penicillin/streptomycin). Cells were stimulated with three peptide poolsfor the Ebola glycoprotein (2.5 μg/mL) for 18 h. Spots were visualizedwith the AEC substrate (BD Biosciences) and quantified with the ELISpotPlate Reader (AID Cell Technology, Strassberg, Germany).

Intracellular Cytokine Staining

PBMCs were isolated from whole blood collected prior to challenge asdescribed (Qiu et al., 2013). The frequency of CD8+ and CD4+ T cellsproducing IFN-γ, IL-2, IL-4, and CD107a were assessed by flow cytometrywith the following antibodies: CD3 Alexa Fluor 700 (clone SP34-2) andCD4 Peridinin Chlorophyll Protein (PerCP)-Cy5.5 (clone L200) from BDBiosciences (San Jose, Calif.), CD8 phycoerythrin (PE)-Cy7 (cloneRPA-T8), CD107a Brilliant Violet 421 (clone H4A3), IL-2 Alexa Fluor 488(clone MQI-17h12), IL-4 PE (clone 8D4-8), and IFN-γ Allophycocyanin(APC, clone B27) from BioLegend (San Diego, Calif.). One million PBMCswere stimulated overnight with peptides (5 μg/mL) using GolgiPlug (0.5μL/mL) and GolgiStop (0.6 μL/mL) in the presence of the anti-CD107aantibody. After surface staining for CD3, CD4, and CD8, samples wereincubated two times (30 min each) in Cytofix/Cytoperm (BD Biosciences)for permeabilization. Intracellular staining was performed, and thesamples were kept overnight in PBS/1° % paraformaldehyde. Approximately250,000-500,000 events were captured on a BD LSR II flow cytometer anddata analyzed with FlowJo vX0.6 software (Tree Star, Ashland, Oreg.).

Measurement of Proliferative Responses by Ki-67 Staining

Blood was collected from each primate in EDTA tubes, shipped same dayand PBMCs isolated as described previously (Hokey et al., 2008). Cellswere resuspended in R10 medium (RPMI 1640, 2 mM 1-glutamine, 50 μMβ-mercaptoethanol, 10% FBS, and 100 IU/mL penicillin and streptomycin)and stimulated using either an Ebola glycoprotein-specific peptidelibrary (2.5 μg/mL), a first generation adenovirus that is geneticallyidentical to the vaccine but does not contain a transgene cassette(AdNull, 1,000 MOI), (22) or 5 μg/mL ConA (Sigma, St. Louis, Mo.) for 5days in 5% CO2 at 37° C. After 3 days, cells were fed by removing 50 μLof spent medium and replacing it with 100 μL of fresh R10 medium. On day5, cells were washed with phosphate buffered saline (PBS) for subsequentimmunostaining for cell surface markers and for Ki-67, an intracellularmarker for proliferation as described (Shedlock et al., 2010).Proliferation was calculated by subtraction of values obtained fromcells cultured in medium alone.

Anti-Ebola Glycoprotein Antibody ELISA

Flat bottom, Immulon 2HB plates (Fisher Scientific, Pittsburgh, Pa.)were coated with purified Ebola virus GP33-637 ΔTM-HA (3 μg/well) in PBS(pH 7.4) overnight at 4° C. (Lee et al., 2009). Heat-inactivated serumsamples were diluted (1:20) in saline. One hundred microliters of eachdilution were added to antigen-coated plates for 2 h at roomtemperature. Plates were washed 4 times and incubated with aHRP-conjugated goat anti-monkey IgG antibody (1:2,000, KPL, Inc.,Gaithersburg, Md.) for 1 h at room temperature. Plates were washed andsubstrate solution added to each well. Optical densities were read at450 nm on a microplate reader (Tecan USA, Research Triangle Park, N.C.).

Neutralizing Antibody Assays

Ebola Virus

Primate sera were heat inactivated at 56° C. for 45 min and thenserially diluted in 2-fold increments in Dulbecco's modified Eagle'smedium (DMEM) in triplicate prior to incubation at 37° C. for 1 h withan equal volume of medium containing EBOV-eGFP (100 PFU per well) asdescribed (Qiu et al., 2012). Virus-serum mixtures were then added toVero E6 cells and placed at 37° C. for 2 days and then fixed in 100%phosphate buffered formalin. GFP levels were quantified by a fluorescentplate reader (AID Cell Technology). These assays were performed underBSL-4 conditions at the National Microbiology Laboratory in Winnipeg.

Adenovirus

Primate sera were heat inactivated and serially diluted as described forthe Ebola virus assay. Samples were incubated with a first generationadenovirus serotype 5 expressing beta-galactosidase for 1 h before theywere added to HeLa cell monolayers. An equal volume of medium containing20% FBS was then added to each well, and infections continued for 24 h.Cells were then histochemically stained for beta-galactosidaseexpression as described (Choi et al., 2012). Positive cells werequantified by visual inspection with a Lecia DM LB microscope (LeicaMicrosystems Inc., Bannockburn, Ill.). For both assays, the serumdilution that corresponded to a 50% reduction in transgene expressionwas calculated by the method of Reed and Muench and reported as thereciprocal of this dilution (Reed et al., 1938).

Quantification of Virus Genomes by Real Time PCR

Ebola Virus

Total RNA was extracted from whole blood using a QIAmp Viral RNA MiniKit (Qiagen). Ebola virus RNA was detected by a qRT-PCR assay targetingthe RNA polymerase (nucleotides 16472 to 16538, AF086833) andLightCycler 480 RNA Master Hydrolysis Probes (Roche Diagnostics GmbH,Mannheim, Germany). The reaction conditions were as follows: 63° C. for3 min, 95° C. for 30 s, and cycling of 95° C. for 15 s, 60° C. for 30 sfor 45 cycles with a LightCycler 480 II (Roche). Primer sequences forthis assay were as follows: EBOVLF2 CAGCCAGCAATTTCTTCCAT (SEQ ID NO: 3),EBOVLR2 TTTCGGTTGCTGTTTCTGTG (SEQ ID NO: 4), and EBOVLP2FAMFAM-ATCATTGGCGTACTGGAGGAGCAG-BHQ1 (SEQ ID NO: 5).

Adenovirus

Urine and BAL fluid were concentrated using Amicon Ultra 100KCentrifugal Filter Devices (Millipore, Billerica, Mass.). DNA wasisolated from blood, concentrated BAL, and oral and nasal swabs using aQIAmp DNA Mini kit according to the manufacturer's instructions (Qiagen,Valencia, Calif.). DNA was isolated from rectal swabs using a modifiedprotocol and the QIAmp DNA Mini kit. DNA was extracted from the urineconcentrate using a QIAamp Viral RNA mini kit (Qiagen) according to themanufacturer's instructions. DNA was isolated from stool samples using aQIAamp Fast DNA Stool Mini kit (Qiagen). Quantification of viral DNA wasdetermined by real time PCR according to a published protocol (Callahanet al., 2006). DNA amplifications were carried out using a ViiA 7Real-Time PCR System (Life Technologies, Carlsbad, Calif.) with thefollowing cycling conditions: 50° C. for 2 min, 95° C. for 10 min, 95°C. for 15 s, and 62° C. for 1 min for a total of 41 cycles. Primersequences, used to amplify a region of the adenovirus serotype 5 hexonprotein, were 5′-ACT ATA TGG ACA ACG TCA ACC CAT T-3′ (forward; SEQ IDNO: 6) and 5′-ACC TTC TGA GGC ACC TGG ATG T-3′ (reverse; SEQ ID NO: 7).The internal probe sequence, tagged with 6FAM fluorescence dye at the 5′end and TAMRA quencher at the 3′ end, was 5′-ACC ACC GCA ATG CTG GCCTGC-3′ (SEQ ID NO: 8). Each sample was run in triplicate in a given PCRassay.

Example 10 Results of Primate Study 1

The first primate study, referred to as Primate Study 1, involved 9 malecynomolgus macaques and served to identify suitable doses of vaccinethat were semiprotective for further evaluation of test formulations toimprove survival in the NHP model. The workflow and treatment schedulesfor the study are depicted in FIGS. 10 and 16A-16B.

Administration of the vaccine at a dose of 1.4×10⁹ infectious virusparticles (ivp)/kg to the respiratory and the sublingual mucosa was welltolerated with no adverse reactions noted. Of particular note is thatall animals experienced a transient increase in serum phosphate levels 6h after immunization with a primate from each treatment group fallingoutside normal values (22473, IM, 1.4 times normal, 50459, IN/IT, 1.2times normal, 62125, SL, 1.3 times normal, FIG. 11A). Phosphate levelsfor all animals reached their nadir at the 24 h time point and werewithin the normal range for the remainder of the study. Blood ureanitrogen (BUN) levels peaked for all animals 24 h after immunization.Two of these animals, one given the vaccine by IM injection (40347, 29mg/dL) and another given the vaccine by the IN/IT route (52945, 33mg/dL), had levels that were notably outside of the normal range (FIG.11B). These values returned to normal by 48 h and remained so throughoutthe course of the study. Serum aspartate aminotransferase (AST), astandard indicator of adenovirus toxicity, (29) was significantlyelevated above normal values in all animals 24 h after immunizationexcept for one animal given the vaccine by the SL route (62125) andanother given the vaccine by the IM route (40347). AST levels fell 48 hafter immunization with only a few animals remaining above normal limits(FIG. 11C). AST values for all animals were within normal limits by the7 day time point. Serum alkaline phosphatase (ALP) of two animals felloutside the normal range during the study. Samples from one animal giventhe vaccine by IM injection were only mildly over the normal acceptablelimit (22473, FIG. 11D) while those of an animal immunized by the IN/ITroute (52945) were 2 times the normal acceptable limit. In both cases,this parameter was high throughout the study and this elevation was notin response to the vaccine. Other serological parameters evaluatedduring the first week after immunization (calcium, creatinine, albumin,globulin, total protein, total bilirubin, alanine aminotransferase(ALT), glucose, sodium, potassium, chloride, and cholesterol) all fellwithin normal limits during the course of the study.

Adenovirus shedding was also evaluated using a standard real time PCRassay to detect adenovirus genomes(28) in serum, nasal swabs, BAL fluid,oral swabs, urine, and feces (FIGS. 12A-12F). A significant number ofadenovirus genomes were found in the serum of one animal immunized bythe respiratory route 2 days after immunization (50459, 1,452 genomes/mLserum, FIG. 12A) and another immunized by IM injection 7 days aftertreatment (22473, 7,296 genomes/mL serum). As expected, substantialamounts of adenovirus serotype 5 genomes were found in nasal swabsobtained from primates immunized by the IN/IT route (50459, 4.2×106,52483, 1.4×106, 59245, 7.5×105) 24 h after immunization (FIG. 12B).Swabs from one primate immunized by the SL route also contained anotable amount of Ad5 genomes (62361, 8,090) at the 24 h time point.Swabs from one animal immunized by the IN/IT route contained asignificant amount of adenovirus genomes 2 days after immunization(52945, 6,333). Samples taken at days 7 and 20 fell below detectionlimits of the assay. Very low amounts of Ad5 genomes were found in theBAL fluid of animals immunized by the IN/IT route 20 days afterimmunization (FIG. 12C). Oral swabs taken 24 h after treatment from oneNHP immunized by the IN/IT route (52483, 4.5×10⁴, FIG. 12D) and twoanimals immunized by the SL route (62125, 4.9×10⁴, and 62361, 9.3×10⁴)contained significant numbers of adenovirus genomes. Swabs collectedfrom animals at the 2 day time point did not contain any adenovirusgenomes. A significant number of virus genomes were detected in theurine of 2 animals within 6 h after treatment (52945, 621 copies/mL, and62361, 1,228 copies/mL). Adenovirus DNA was also found 24 h aftertreatment in the urine of 3 animals (50459, 1,163, 62361, 801, and62125, 116 copies/mL, FIG. 12E). Samples from all other animalsthroughout the time course of this study fell below detection limits ofthe assay. Interestingly, adenovirus genomes were only detected in thefeces of animals immunized by the IN/IT route (FIG. 12F). As early as 6h after immunization, 2.362 and 7,302 adenovirus genomes were found infecal samples from animals 52483 and 52945 respectively. Feces collectedfrom animal 50459 24 h after vaccination contained 5,919 adenovirusgenomes. This increased to 7,405 in samples taken from the same animalat the 48 h time point. Samples from animal 52945 also taken 48 h aftertreatment contained 2,772 virus genomes.

The T Cell Response

Twenty days after immunization, PBMCs were isolated from whole blood andincubated with peptides specific for Ebola glycoprotein (GP). Cells werethen subjected to intracellular cytokine staining for CD8+ and CD4+surface antigens and IFN-γ and sorted by flow cytometry. At this timepoint, few cells responsive to Ebola glycoprotein could be detected inPBMCs obtained from any of the animals (data not shown). A similar trendwas observed in samples taken from iliac lymph nodes (ILNs) of animals.Profound responses were seen in samples obtained from the BAL fluid ofanimals given the vaccine by the IN/IT route. The strongest response wasseen in CD4+ cells with 12.5% of the population obtained from primate52945 and 3.03% of the population from primate 50459 responding (FIG.13A). Although the response from the third primate in this treatmentgroup (52483) was small in comparison (0.71%), it was significantlyhigher than that observed in animals given the vaccine by IM injection.The CD8+ T cell response followed a similar trend (FIG. 13B).

PBMC and ILN populations were further analyzed for IFN-γ production inresponse to Ebola GP by ELISpot. Samples from animals immunized by theIM route (22473 and 40347) both had significant numbers of IFN-γproducing cells (255 and 642 spot forming cells (SFCs)/millionmononuclear cells (MNCs) respectively. FIG. 13C). PBMC samples from twoNHPs immunized by the SL route (52165, 62361) also had measurablenumbers of IFN-γ producing cells (257 and 98 SFCs/million MNCs). Samplesfrom NHPs immunized by the IN/IT route contained the highest numbers ofIFN-γ producing cells (1,100, 607, and 2,055 SFCs/million MNCs). Samplesfrom the ILNs of 2 NHPs given the vaccine by the IN/IT route (50459 and52945) contained approximately 7 and 18 times the number of IFN-7producing cells found in the saline control (animal 22457) respectively(FIG. 13D).

38 days after immunization, the proliferative capacity of CD4+ and CD8+cells in response to Ebola GP and adenovirus serotype 5 was assessed bya Ki-67 staining assay (Shedlock et al., 2010). Two samples, eachobtained from animals immunized by the respiratory route, containedsignificant numbers of proliferative Ebola GP-specific CD4+ T cells(50459, 11.9%, and 52945, 6.5%, white bars, FIG. 13E). The sampleobtained from NHP 50459 also contained the most Ebola GP-specific CD8+ Tcells (8.8%, black bars, FIG. 13E). The sample from NHP 62125 immunizedby the SL route contained the second highest amount of CD8+ T cells(4.9%). All remaining samples contained approximately 3-4% CD8+ T cellsthat could proliferate in response to Ebola GP except for that fromanimal 52483 (1.1%). Only one sample obtained from a primate immunizedby the IN/IT route, 52165, contained a significant population ofproliferative adenovirus 5-specific CD4+ T cells (8.1%, white bars, FIG.13F). One sample from a primate in the IN/IT group (50459) and anotherfrom the SL group (62125) contained notable populations of CD8+ cellsthat proliferated in response to Ad5 (9.4 and 9.3% respectively, blackbars, FIG. 13F). All remaining samples contained approximately 4% CD8+ Tcells that could proliferate in response to adenovirus except for animal40347 (2.2%).

The Antibody-Mediated Response

Anti-Ebola GP and anti-adenovirus antibody levels were assessed in serumand BAL fluid 20 and 38 days after immunization (FIGS. 14A-14C). Markedlevels of anti-Ebola GP IgG antibodies were found in serum from animalsimmunized by the IM and the IN/IT routes 20 days after treatment (FIG.14A). These levels increased further 38 days after vaccination.Anti-Ebola GP antibodies were found in the serum of only one of theanimals immunized by the SL route (52165). This animal also had EbolaGP-specific IgG antibodies in BAL fluid 20 days after treatment (FIG.14B) that were similar to those found in samples from animals immunizedby the respiratory route. BAL from animals immunized by the IM route didnot contain any detectable levels of anti-Ebola GP antibodies. Onesample from a primate immunized by the IM route (40347) contained asignificant amount of circulating anti-adenovirus neutralizingantibodies (NABs, 1,007 reciprocal dilution, FIG. 14C). The sample fromthe remaining animal in the IM group and 2 others from the IN/IT groupcontained anti-adenovirus NAB titers of ˜200 reciprocal dilution. Serumfrom animals immunized by the SL route did not contain measurable levelsof anti-adenovirus 5 NABs.

Lethal Challenge with Ebola Virus

62 days after immunization, NHPs were challenged with 1,000 pfu of Ebolavirus (1995, Kikwit). One primate immunized by IM injection (40347) andone animal immunized by the SL route (62125) succumbed to infection 6days after challenge (FIG. 15A). At this time animal 62125 had aclinical score of 23, and substantial petechiae were noted uponnecropsy. Primate 40347 had a temperature of 40.3° C. and a clinicalscore of 25 and experienced notable bleeding. One primate immunized bythe IN/IT route (52483) and one primate immunized by the SL route(62361) died the following day. Each of these animals had clinicalscores above 25 and significantly decreased food intake the previousday. The remaining primate immunized by the SL route (52165) expired 8days after challenge. One of the primates vaccinated by IM injection(22473) and two of the animals immunized by the IN/IT route (50459,52945) survived challenge (50 and 67% survival IM and IN/ITrespectively, FIG. 15A). Moderate drops in body weight were noted duringinfection (FIG. 15B). A slight increase in weight of one animalimmunized by the IN/IT route (50459) was noted during the study period.Changes in body temperature (FIG. 15C) and clinical scores (FIG. 15D)for each primate were in line with survival results. The most strikingchanges in hematology and blood chemistry values were observed aroundday 5 postchallenge in the animals that did not survive. These includesignificantly elevated liver enzymes with ALT (FIG. 15E) and ALP (FIG.15F) values rising to levels 27 and 16 times baseline respectively andblood urea nitrogen levels rising to 7.5 times normal values before theanimals expired (FIG. 15G). Platelet counts, however, dropped to halfthe baseline values in these animals (FIG. 15H). In contrast, a sharpincrease in platelets was noted in samples obtained from animals thatsurvived challenge. Other hematology and blood chemistry values in theseanimals remained largely unchanged (data not shown).

TABLE 3 Primate Study 2: Shedding Patterns of Adenovirus DNA from theRectal Mucosa of Non-Human Primates after a Single Dose ofAdCAGoptZGP^(a) route of immunization animal # pre 0.25 d 1 d 2 d 7 d 20d IN/IT 0810003  —^(b)   1,500^(c)  10 × 10⁵ 3.7 × 10⁴ 2,000 2,1000802197 —   380 7.5 × 10⁵ 2.6 × 10^(s) 420 540 0809077 — — 3.1 × 10⁴ 9.0× 10⁴ 620 2,600 SL 0805257 —   83 6.0 × 10⁶ 3.6 × 10⁵ 780 79 0804317 —1,400 3.5 × 10⁴ 1.5 × 10⁴ 640 58 0808233 —   200 5,600 1,600 5,600 24PEI-SL 0807243 — 1,100 1.2 × 10⁵ 1,100 190 58 0809227 —   920   440 1.1× 10⁴ 130 — 0804819 — 2,000 1.4 × 10⁶ 1,900 30 — ^(a)Data were obtainedby real-time TaqMan PCR on DNA isolated from samples as described.^(b)None detected. Sample fell below the detection limit of the assay(10 viral genomes/100 ng of DNA). ^(c)Units are genome copies per swab.

Example 11 Results of Primate Study 2

The second study, referred to as Primate Study 2, evaluated a novelformulation for the respiratory platform and involved refinement of thesublingual platform in naive animals and those with prior exposure toadenovirus. The workflow and treatment schedules for the study aredepicted in FIGS. 10 and 16A-16B.

Effect of Formulation on Establishing Long-Lasting Immunity to Ebola andRefinement of Dose for Sublingual Immunization

The most exciting finding extracted from Study 1 was that the combinedIN/IT administration of the vaccine was able to confer long-termimmunity to Ebola. Since it was not known if immunity induced byadenovirus-based vaccines for Ebola is persistent over time (Vasconceloset al., 2012; Majhen et al., 2014), it was decided to extend the lengthof time between respiratory administration of a formulated version ofthe vaccine and challenge. A secondary goal was to increase the dose ofvaccine given by the sublingual route and to evaluate the ability of thesublingual vaccine to confer protection in animals with prior exposureto adenovirus since improved responses in this population were observedin studies with rodents (Choi et al., 2013). The long-term immuneresponse of surviving animals postchallenge was also a major point ofinterest in this study especially in animals receiving vaccinecontaining a novel formulation (Choi et al., 2014) and in those giventhe sublingual vaccine to identify parameters to target duringadditional refinement of each immunization platform.

Three male cynomolgus macaques were given the vaccine in a potassiumphosphate buffer (pH 7.4) containing an amphiphilic polymer (formulaweight (FW) ˜39,000) formulation that improved the antigen-specificimmune response in rodent models of infection (Choi et al., 2014). Thegoal was to immunize this group as early in the study as possible sothat there would be a significant amount of time between immunizationand challenge (FIGS. 16A-16B). 42 days after these animals wereimmunized, 3 macaques were given 1×1011 particles of a host range mutantadenovirus serotype 5 that can replicate in non-human primates (Buge etal., 1997; Klessing et al., 1979) by intramuscular injection toestablish pre-existing immunity. 42 days later, animals were then giventhe vaccine by the sublingual route. At this time the animals had anaverage circulating anti-adenovirus antibody titer of 320±160 reciprocaldilution. Three naive animals were also given the same dose of vaccineby the sublingual route at the same time for comparison.

Toxicology and Vaccine Shedding

In contrast to the first study, a notable spike in creatinephosphokinase (CPK) was detected in the serum of all animals 24 h afterimmunization (FIG. 17A). This enzyme increased to 8 times baseline inone animal immunized by the IN/IT route (810003, 8,209 IU/L) and to 10times baseline in a primate with pre-existing immunity to adenovirusimmunized by the sublingual route (804819, 4,483). A notable spike inserum lactate dehydrogenase (LDH) was also noted at the 24 h time point.This was not as sharp as that seen with CPK with the highest elevationsfound to be approximately 3 times baseline (804317, 849 IU/L, FIG. 17B).Both parameters returned to normal within 3 days after treatment. Asseen in the first study, serum AST increased in all primates afterimmunization. This occurred at the 24 h time point for animals immunizedby the respiratory and sublingual routes but was not observed inprimates with pre-existing immunity to adenovirus until 48 h (FIG. 17C).As in the first study, serum alkaline phosphatase (ALP) levels variedbetween primates, however, in this trial a distinct drop in thisparameter was noted in samples collected from most animals between the 6and 24 h time points, after which values remained constant (FIG. 17D).Other serological parameters evaluated during the first week afterimmunization (calcium, creatinine, albumin, globulin, total protein,gamma glutamyl transferase (GGT), total bilirubin, alanineaminotransferase (ALT), BUN, glucose, sodium, potassium, phosphate,chloride, and cholesterol) all fell within normal limits throughout thecourse of the study (data not shown).

Adenovirus genomes were only found in serum samples collected fromanimals immunized by the respiratory route (FIG. 18A). The mostsignificant numbers of virus genomes detected in any of the biologicalsamples collected throughout the second study were found in nasal swabscollected from primates 6 h after IN/IT immunization [810003 (8.18×106genome copies (GC)), 809077 (1.44×107 GC), and 802197 (1.36×107 GC, FIG.18B)] and in oral swabs collected from primates 6 h after sublingualimmunization: [804317 (9.06×106 GC), 805257 (1.74×105 GC), and 808233(7.92×10⁶ GC, FIG. 18D)]. As seen in the first study, adenovirus genomeswere only found in the BAL fluid of animals immunized by the IN/IT route(FIG. 18C). Urine collected from one naive animal immunized by the SLroute and another with pre-existing immunity also immunized by the SLroute 6 h after treatment contained notable amounts of adenovirus(808233, 9,821 GC; 807243, 2,363 GC, FIG. 18E). Adenovirus genomes werefound in feces collected from one primate with pre-existing immunity toadenovirus 24 h after immunization by the SL route (804819, 2.71×106 GC)and in another primate 2 days after it was immunized by the IN/IT route(802197, 6.51×106 GC, FIG. 18F). Virus continued to be shed in feces ofthis animal 1 week after immunization (802197, 2.52×106 GC). AdenovirusDNA was found on rectal swabs collected from each animal throughout thecourse of the study (Table 3).

The Long-Term T Cell Response

The Ebola virus glycoprotein-specific T cell response was examined inPBMCs isolated from whole blood immediately prior to challenge, 150 dayspostimmunization. Multiparameter flow cytometry provided a comprehensiveanalysis of the types of antigen-specific T cells elicited by eachtreatment (FIGS. 19A-19D). The CD4⁺ T cell population present in animalsimmunized by the IN/IT route was much more diverse than the CD8⁺ T cellpopulation (FIGS. 19A and 19B). Six specific CD4⁺ T cell subpopulationswere found in animal 802197 with the most predominate phenotype beingCD4⁺ CD107a⁺ IL-2⁺ (39% of the CD4⁺ population, FIG. 19A). This animalalso had the most diverse antigen-specific CD8⁺ T cell population with 4different subpopulations detected by intracellular staining (FIG. 19B).Samples from NHP 809077 contained four different CD4⁺ subpopulations.Cells that were CD4⁺ IL-2⁺ were most prevalent (45%) in this primate.The CD8⁺ population in this animal was composed of 3 specific subtypeswith relatively equal distribution (CD8⁺ CD107a⁺ IL-2⁺, CD8⁺ IFN-γ⁺, andCD8⁺ IL-2⁺). The CD4⁺ T cell population was less diverse in primate810003 with the majority of antigen-specific cells also having the CD4⁺IL-2⁺ phenotype (85%). The CD8⁺ IL-2⁺ subpopulation was the mostprominent of two types of antigen-specific CD8⁺ T cells found in thisprimate.

CD4⁺ and CD8⁺ T cell populations were noticeably less diverse in animalsimmunized by the SL route (FIG. 19C). Antigen-specific CD4⁺ T cells werenot detected in samples collected from primate 808233. CD4⁺ IFN-γ⁺ IL-2⁺cells were present to a lesser degree than CD4⁺ IFN-γ⁺ cells in samplescollected from animal 805257 (25% and 75% of the populationrespectively). The most diverse CD4⁺ population elicited by SLimmunization was found in primate 804317 with CD4⁺ IL-2⁺ cells being themost prominent of 5 different subtypes identified in this population.Antigen-specific CD8⁺ T cells were only found in samples collected fromthis animal with the majority being of the CD8⁺ CD107a⁺ phenotype(92.6%) and the remaining cells of the CD8⁺ IL-2⁺; phenotype (7.4%, datanot shown).

Pre-existing immunity to adenovirus did not noticeably alter thediversity of T cells elicited by sublingual immunization (FIG. 19D).Five distinct subpopulations of CD4⁺ T cells were found in primate809227 with those of the CD4⁺ IL-2⁺ being the most prominent (63.1%). Asingle population of CD8⁺ CD107a⁺ cells was also found in samplescollected from this animal (data not shown). CD4⁺ IL-4⁺ cells were themost prominent of the two antigen-specific CD4⁺ T cell populations foundin samples collected from primate 807243. Antigen-specific CD8⁺ T cellswere not detected in samples collected from this animal. SL immunizationinduced a single population of CD4⁺ IL-2⁺ cells and a single populationof CD8⁺ CD107a⁺ cells in primate 804819.

The Antibody-Mediated Response

Anti-Ebola GP and anti-adenovirus antibody levels were assessed in serumand BAL fluid at various time points after immunization (FIGS. 20A-20F).Antigen-specific antibody levels mildly increased between day 20 and day104 in serum collected from two animals immunized by the IN/IT route(0810003, 1.5-fold increase, 0809077, 1.3-fold increase, 0802197, nochange, FIG. 20A). Antibody levels remained high at the 142 day timepoint and were comparable to those found in animals immunized by therespiratory route in the first primate study. Significant anti-Ebola GPantibody levels were detected in the BAL fluid of only one primateimmunized by the IN/IT route (0802197, FIG. 20B). Samples obtained fromone of the animals immunized by the sublingual route (0808233) containedthe highest level of anti-Ebola GP antibodies than any of the otheranimals given a single dose of vaccine (FIG. 20C). It is also importantto note that a significant change in anti-Ebola GP antibody levelsbetween day 20 and day 57 postimmunization was detected in samplesobtained from only one animal in this treatment group (0805257, 2.4-foldincrease). Samples from only one of the animals with prior exposure toadenovirus immunized by the sublingual route contained anti-Ebola GPantibodies above the detection limit of the assay (809227, FIG. 20D).While a notable amount of anti-adenovirus neutralizing antibody (NAB)was detected in the serum of one primate 20 days after immunization bythe IN/IT route (802197, 1:640 reciprocal dilution), circulatinganti-adenovirus NABs were low in samples obtained from other primatesimmunized in the same manner (FIG. 20E). Anti-adenovirus NABs were notfound in the BAL of any of the primates immunized by the IN/IT routeduring the course of the study (data not shown). While anti-adenovirusNABs were quite high in the serum of one animal with pre-existingimmunity 20 days after immunization by the SL route (809227, 1:2,560reciprocal dilution), they were not detected in samples collected fromtwo naive primates immunized in the same manner (805257, 804317, FIG.20F).

Lethal Challenge with Ebola Virus

150 days after immunization, animals were challenged with 1,000 pfu ofEbola virus (1995, Kikwit). Six days after challenge, both primatesgiven saline, two animals immunized by the SL route (804317, 808233),and one animal with pre-existing immunity to adenovirus immunized by theSL route (809227) expired from infection (FIG. 21A). The remainingprimates with pre-existing immunity succumbed to infection on days 7(804819) and 8 (807243) respectively. The remaining animal given thevaccine by the SL route (805257) expired on day 9. Each animal immunizedby the respiratory route survived challenge. These animals experiencedminimal changes in body weight (FIG. 21B) and temperature (FIG. 21C)during the course of infection with their clinical scores peaking atabout 4-7 days after challenge (FIG. 21D).

A notable drop in lymphocyte levels of all animals was observed 3 daysafter challenge (FIG. 21E). Lymphocytes abruptly spiked in one animalimmunized by the SL route (808233) and another with pre-existingimmunity to adenovirus (804819) 6 days after challenge. Lymphocytelevels of primates immunized by the IN/IT route slowly increased to day14 where they remained constant. Lymphocytes of all other animalsremained low until the time of death. ELISpot analysis revealed that asignificant amount of MNCs capable of producing IFN-γ in response tostimulation with Ebola GP peptides were present in PBMCs isolated fromwhole blood of surviving animals 14 days after challenge (FIG. 21F). Asharp drop in platelet counts was noted in all animals that did notsurvive challenge (FIG. 21G). Mild drops in platelet counts wereobserved in animals immunized by the IN/IT route 3 days after challenge.These values continued to drop through day 28. ALT (FIG. 21H) and BUN(FIG. 21I) sharply rose to values as high as 24 and 6 times baselinerespectively in animals that succumbed to Ebola infection. These valuesremained unchanged throughout Ebola infection in surviving animals.

Assessment of sera taken during challenge revealed that primatesimmunized by the IN/IT route had very high levels of circulatinganti-Ebola GP antibodies (FIG. 22A). These were neutralizing since verylow levels of infectious Ebola were found in samples taken from twoprimates 3 days postchallenge (FIG. 22B). Infectious Ebola virus was notdetected in any samples collected from the third animal in thistreatment group (809077). Ebola virus genomes were also not detected insamples taken from any of the animals immunized by the respiratory route(Table 4). Although samples from two animals immunized by the sublingualroute also contained high levels of anti-Ebola neutralizing antibody(804317, 808233, 1,280 reciprocal dilution, FIG. 22C), they were onlypartially neutralizing since a concentration of 316 TCID₅₀/mL was foundin samples collected from both primates at the 3 day time point thatescalated to 1.47×10⁸ and 6.81×10⁸ TCID50/mL respectively by the 6 daytime point (FIG. 22D). The number of circulating virus genomes in theseanimals followed a similar trend (Table 4). One animal that was exposedto the adenovirus serotype 5 host range mutant prior to immunization bythe SL route (804819) also had high levels of anti-Ebola GP circulatingantibodies (1,280 reciprocal dilution, FIG. 22E), however, Ebola virusRNA was detected in samples collected from this animal at aconcentration of 8.19×10⁶ genome copies/mL (Table 4). This animalexpired before any infectious virus could be detected in its serum (FIG.22F).

TABLE 4 Primate Study 2: Circulating Ebola Virus Genomes in PrimatesChallenged with Ebola Virus 150 Days after Immunization with a SingleDose of AdCAGoptZGP^(a). animal no. treatment/route day 0 day 3 day 3.8day 14 day 21 day 28 0810091 KPBS  —^(b) 880^(c) 1.84 × 10⁵ d  N.A.^(e)N.A. 0805201 KPBS — — 7.79 × 10⁵ d N.A. N.A. 0802197 IN/IT — — N.A. — —— 0809077 IN/IT — — N.A. — — — 0810003 IN/IT — — N.A. — — — 0805257 SL —— N.A. d N.A. N.A. 0804317 SL — 1.74 × 10⁴ 9.84 × 10⁶ d N.A. N.A.0808233 SL — 1.01 × 10³ 1.14 × 10⁶ d N.A. N.A. 0809227 PEI-SL — 2.08 ×10⁴ 1.57 × 10⁴ d N.A. N.A. 0804819 PEI-SL — — 8.19 × 10⁶ d N.A. N.A.0807243 PEI-SL — 3.33 × 10⁴  3.3 × 10⁵ d N.A. N.A. ^(a)Data wereobtained by quantitative RT-PCR on RNA isolated from whole blood asdescribed. ^(b)None detected. Sample fell below the detection limit ofthe assay (86 viral genomes/mL). ^(c)Units are genome copies permilliliter of whole blood (GC/mL). d - Animal expired prior to samplecollection at this time point. ^(e)Not assayed at this time point.

Example 12 Flexible Film Technology

Biologicals can be stabilized in small, unit dose films useful foradministration to a variety of animal models and for evaluation oflong-term stability of vaccines during long-term storage (see FIG. 23A).Several thousand doses of a given biological substance can be stabilizedin large films that can be divided into single-use doses (see FIG. 23B).

A supersaturated solution was created by adding sufficient stabilizers(sugars and sugar derivatives, polymers) and permeability enhancers(surfactants) to a solvent system (distilled deionized water, trisbuffer, ethanol, methanol) such that the total amount of solidcomponents added to the solvent were within the concentration of 10-90%w/w. In some embodiments, the solution was formulated comprisingpotassium phosphate buffered saline (pH 7.4), a detergent (10 mg/mlPMAL-C16 in certain aspects) and, optionally, one or more sugars.

This suspension was prepared by stirring, homogenization, mixing and/orblending these compounds with the solvent. Small portions of eachcomponent (˜ 1/10 the total amount) were added to the solvent and thesolution was mixed before adding additional portions of the same agentor a new agent.

Once each stabilizer and permeability enhancer was added, the bulksolution was placed at 4° C. for a period of time between 2-24 hours.After this time, the bulk solution was subject to sonication for aperiod of 5-120 minutes to remove trapped air bubbles in thepreparation.

After sonication was complete, the recombinant adenovirus vector wasadded to the preparation. The amount of adenovirus ranged from of0.1-300% of the total solid concentration. Adjuvants, in some aspects,were added at this time. The amount of these compounds ranged from0.005-10% of the total solid concentration. These agents were added bygentle stirring (10-50 rpm) so as to not induce air pockets and bubbleformation in the final preparation.

The preparation was then slowly piped into molds of a shape suitable forthe application. In certain aspects, the molds can be constructed ofstainless steel, glass, silicone, polystyrene, polypropylene and otherpharmaceutical grade plastics. In some aspects, the preparation can beplaced in the molds by slowly pouring by hand or by pushing thepreparation through a narrow opening on a collective container at a slowcontrolled rate (0.25 ml/min) to prevent early hardening and/or bubbleformation in the final film product. Films were poured to a thickness of12.5-1000 μm.

Molds for casting of films were sterilized by autoclaving and placed inlaminar air flow hoods prior to casting. Molds were also sometimes linedwith a peelable backing material suitable for protection of the filmproduct. Suitable backings can be made of aluminum, gelatin, polyesters,polyethylene, polyvinyl and poly lactic co-glycolide polymers and/or anyother pharmaceutically acceptable plastic polymer.

Cast films remained at ambient temperature (20-25° C.) in a laminar flowhood for 2-24 hours after which time a thin, peelable film was formed.Some films were opaque or, preferably, translucent (see FIGS. 23A-23B).Films were then stored at room temperature under controlled humidityconditions. Some films were stored at 4° C. under controlled humidity aswell.

Films were porous, amorphous solids that stabilized the recombinantadenovirus vector in their native three-dimensional shape (see FIGS.24A-24D). The films retained the shape of the embedded virus aftertwelve months of storage at room temperature (FIG. 25).

Multilayer films can also be created at this time by applying a secondcoating of a supersaturated solution containing the same antigen as thefirst layer or another different adjuvant/antigen system to the thinfilm in a laminar flow hood. This will remain at ambient temperature(20-25° C.) in a laminar flow hood for an additional 2-24 hours afterwhich time a thin, peelable film will be formed. This film may beopaque. It may also be translucent. In certain aspects the films maylikewise comprise multiple film layers.

Films were dissolved in saline or simulated human saliva warmed to 37°C. (body temperature) and time needed for full dissolution notedimmediately upon drying and at various times during storage. Theresulting solutions were screened for antigen confirmation and activityto determine the effectiveness of the formulation to retain the potencyof the preparation over time. The results are shown in FIGS. 26-40.

Both infectious enveloped and non-enveloped viruses were found to berecoverable from dried film (FIG. 26). Infectious titers of recombinantadenovirus expressing the Ebola Virus glycoprotein (a non-envelopedvirus) and PR8 (H1N1 influenza) were added to liquid formulations, driedand reconstituted 48 hours after storage in the dry state at 20° C.Percent recovery was between 85% and 95% for both viruses (FIG. 26).

Different solvent systems influenced changes in film pH during thedrying process (FIG. 27). Solvents such as distilled, deionized water(1), PBS (phosphate buffered saline (2)), and Tris(Tris(hydroxymethyl)aminomethane (3)) were used (see FIG. 27). Thesolvent system also dictated recovery of virus from dried film (FIGS.28A-28C). The pH of the dried film significantly impacted recovery ofinfectious virus after reconstitution (FIG. 29). The lower the pH, thelower the percent recovery; when the pH was higher, percent recovery washigher (FIG. 29). The addition of detergent to the formulation was foundto prevent a drop in film pH after drying (FIG. 30).

Base formulations also played a role in recovery of virus from driedfilm. Three different stabilizer concentrations were evaluated for theirability to retain infectious titer of virus after drying in twodifferent solvent systems with varying results (FIGS. 31A-31B). Inaddition to affecting pH levels, detergent was found to significantlyimprove recovery of infectious virus from the films (FIG. 32). However,the amount of virus embedded in film formulation did not impact recovery(FIG. 33). The use of binding agents improved recovery of recombinantvirus film (FIG. 34).

When compared to blank controls, the virus presence was found tosignificantly impact dissolution rate of films in simulated human saliva(FIG. 35). The addition of a detergent to the film then significantlyimproved dissolution time (FIG. 36). Film formulations with and withoutdetergent were shown to protect adenovirus from degradation in saliva(FIG. 37).

The presence of virus in the film significantly increased the moistureretention in dried film formulations when compared to blank controls(FIG. 38). The addition of detergent profoundly increased moisturecontent in dried film containing virus (FIG. 39). Additionally, it wasfound that recombinant adenovirus can be evenly distributed across largefilms that can then be divided into equal unit doses (FIG. 40).

Examples of formulations used in these experiments are shown below inTable 5.

TABLE 5 0.5 = 0.5% (w/w) HPMC, 1.5 = 1.5% (w/w) HPMC, 3 = 3% (w/w) HPMC,2S = 2% (w/w) Sorbitol, 2G = 2% (v/v) Glycerol. All formulationscontained 0.2% w/v tragacanth gum. 1 .5 2 .5/2S 3 .5/2G 4 1.5 5 1.5/2S 61.5/2G 7 3 8 3/2S 9 3/2G

The stability of viral particles in a formulation of the embodiments isshown in FIGS. 41A-41B. As can be seen in FIG. 41B, even when in aliquid form, the adenovirus retained nearly all its starting infectivityafter 8 months of storage. Importantly, when stored in a solidformulation detailed herein nearly all infectivity can be maintained forlonger than 36 months (see FIG. 41A).

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Theparticular embodiments disclosed above are illustrative only, as thepresent invention may be modified and practiced in different butequivalent manners apparent to those skilled in the art having thebenefit of the teachings herein. Furthermore, no limitations areintended to the details of construction or design herein shown, otherthan as described in the claims below. It is therefore evident that theparticular illustrative embodiments disclosed above may be altered ormodified and all such variations are considered within the scope andspirit of the present invention. While compositions and methods aredescribed in terms of “comprising,” “containing,” or “including” variouscomponents or steps, the compositions and methods can also “consistessentially of” or “consist of” the various components and steps. Allnumbers and ranges disclosed above may vary by some amount. Whenever anumerical range with a lower limit and an upper limit is disclosed, anynumber and any included range falling within the range is specificallydisclosed. In particular, every range of values (of the form, “fromabout a to about b,” or, equivalently, “from approximately a to b,” or,equivalently, “from approximately a-b”) disclosed herein is to beunderstood to set forth every number and range encompassed within thebroader range of values. Also, the terms in the claims have their plain,ordinary meaning unless otherwise explicitly and clearly defined by thepatentee. Moreover, the indefinite articles “a” or “an,” as used in theclaims, are defined herein to mean one or more than one of the elementthat it introduces. If there is any conflict in the usages of a word orterm in this specification and one or more patent or other documentsthat may be incorporated herein by reference, the definitions that areconsistent with this specification should be adopted.

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What is claimed is:
 1. A composition comprising a recombinant viralvector comprising an expression cassette encoding a heterologouspolypeptide, said recombinant viral vector formulated in a substantiallysolid carrier comprising: (i) PMAL-C16 or (ii) from about 0.1% to 10% ofa zwitterionic surfactant.
 2. The composition of claim 1, wherein thesubstantially solid carrier comprises less than 5% water.
 3. Thecomposition of claim 1, further comprising a stabilizing agent, whereinthe stabilizing agent is carbohydrate stabilizer selected from thegroups consisting of sucrose, mannitol, melezitose, trehalose or mixturethereof.
 4. The composition of claim 1, further comprising a plasticizeragent, wherein the plasticizer agent is a gum selected from the groupconsisting of guar gum, fenugreek gum, locust bean gum, Gum arabic,karaya gum, Carrageenan, Pullulan, agar-agar, or an alginate.
 5. Thecomposition of claim 1, wherein the carrier comprises PMAL-C16.
 6. Thecomposition of claim 1, wherein the recombinant viral vector is anon-enveloped virus.
 7. The composition of claim 1, wherein therecombinant viral vector is an enveloped virus.
 8. The composition ofclaim 1, wherein the solid amorphous carrier further comprises a pHbuffering agent sufficient to provide a final pH of 5.0-8.0.
 9. A methodfor delivering a recombinant viral vector encoding a heterologouspolypeptide to a mammal, comprising: (a) obtaining a composition inaccordance with claim 1, which has been dispersed in a pharmaceuticallyacceptable liquid; and (b) administering an effective amount of thedispersed composition a mammal.
 10. The method of claim 9 wherein theadministering comprises administering the dispersed composition to amucosal tissue of the mammal.
 11. The method of claim 10, wherein theadministering is by oral, sublingual, buccal or intranasaladministration.
 12. The method of claim 9, wherein the pharmaceuticallyacceptable liquid is distilled deionized water.
 13. The method of claim9, wherein recombinant viral vector is a non-enveloped virus.
 14. Themethod of claim 9, wherein composition of step (a) comprises PMAL-C16.15. The method of claim 9, wherein composition of step (a) comprisesabout 0.1% to 10% of a zwitterionic surfactant.
 16. The method of claim9, wherein the composition dispersed in the liquid comprises about 0.1to 50 mg/ml of the zwitterionic surfactant.
 17. The method of claim 9,wherein the composition dispersed in the liquid comprises about 0.1 to50 mg/ml of PMAL-C16.
 18. The method of claim 9, wherein obtaining thecomposition comprises solubilizing the solid composition in an aqueousliquid.
 19. The method of claim 9, wherein the mammal has beenpreviously exposed to a virus that crossreacts antigenically with therecombinant viral vector of the composition.
 20. A method of making astabilized recombinant viral vector composition comprising: (a)formulating a solution comprising the recombinant viral vectorcomprising an expression cassette encoding a heterologous polypeptide ina pharmaceutically acceptable carrier said carrier comprising: (i)PMAL-C16; or (ii) from about 0.1% to 10% of a zwitterionic surfactant;and (b) drying the solution to provide a stabilized recombinant viralvector composition in a substantially solid amorphous carrier.
 21. Themethod of claim 20, wherein drying the solution comprises dispersing thesolution in a thin film and allowing the liquid to evaporate in theabsence of external pressure or heat.
 22. The method of claim 20,wherein prior to drying the solution comprises about 0.1 to 50 mg/ml ofPMAL-C16.
 23. The method of claim 20, wherein the recombinant viralvector is an enveloped virus.
 24. The method of claim 20, wherein therecombinant viral vector is a non-enveloped virus.